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

Abstract

The invention relates to the technical field of biological materials, in particular to an injectable acellular fat matrix preparation as well as a preparation method and application thereof. The preparation method of the injectable decellularized fat matrix preparation comprises the following steps: taking carbon dioxide as a solvent, and removing cells from disinfected fat tissues by using a supercritical fluid method to obtain a decellularized fat matrix; freezing and grinding the decellularized fat matrix by using liquid nitrogen to obtain decellularized fat matrix powder; and dissolving the decellularized fat matrix powder in the preparation solution, filling and sterilizing. According to the preparation method provided by the embodiment of the invention, the pre-filled injectable acellular fat matrix preparation is obtained, and the preparation has excellent biocompatibility and has a remarkable curative effect on osteoarthritis.

Description

Technical Field

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

[0002] With the continuous development of biomedical technology, the demand for tissue repair and regeneration is growing. Among many biomaterials, decellularized matrix has attracted much attention due to its unique biological properties and clinical application potential. Decellularized matrix can not only provide good physical structural support, but also participate in regulating cell adhesion, migration, proliferation and differentiation. Therefore, it has broad application prospects in the fields of tissue regeneration and repair, promoting vascular differentiation, inducing adipocyte production and neural repair.

[0003] In the related technologies, the preparation methods for decellularized fat matrix have defects such as low extraction efficiency, complicated process, incomplete degreasing, and destruction of the microstructure and effective ingredients of the extracellular matrix. It is not only difficult to carry out standard industrial production, but the obtained products cannot be directly applied in clinical practice, which limits the development of decellularized fat matrix preparations.

[0004] In response to the above-mentioned problems, developing a new and improved extraction and preparation method to improve the extraction efficiency and maintain the intact three-dimensional structure of the extracellular matrix while reducing the waste of raw materials and environmental pollution is an important need in the current field. Summary of the invention

[0005] The present invention solves one of the problems of the related art from at least the following aspects.

[0006] In a first aspect, an embodiment of the present invention provides a method for preparing an injectable decellularized fat matrix preparation, comprising: removing cells from sterilized fat tissue using a supercritical fluid method with carbon dioxide as a solvent to obtain a decellularized fat matrix; freezing and grinding the decellularized fat matrix using liquid nitrogen to obtain a particle size D 90 Decellularized fat matrix powder with a size less than 500 μm; the decellularized fat matrix powder is dissolved in a preparation solution, then filled and sterilized to obtain the injectable decellularized fat matrix preparation.

[0007] In some embodiments, the freezing and grinding of the decellularized fat matrix using liquid nitrogen comprises: grinding the decellularized fat matrix in liquid nitrogen at a speed of 5000 rpm-8000 rpm, for a total of 2-5 times, and each grinding time is 2-5 minutes.

[0008] In some embodiments, the step of dissolving the decellularized adipose matrix powder in a preparation solution and then filling and sterilizing the solution comprises: dissolving the decellularized adipose matrix powder in a preparation solution and then filling the solution into a syringe and sterilizing the syringe.

[0009] In some embodiments, the injectable decellularized adipose matrix preparation comprises 1 to 30 mg / ml, preferably 10 to 30 mg / ml, of the decellularized matrix powder.

[0010] In some embodiments, the method of sterilization is irradiation sterilization or ethylene oxide sterilization.

[0011] In some embodiments, the sterilization method is irradiation sterilization at a radiation dose of 5-40 kGy, preferably 20-30 kGy.

[0012] In some embodiments, the conditions of the supercritical fluid method include: pressure of 20-25 MPa, temperature of 33-37° C., carbon dioxide flow rate of 0.09-0.1 L / min, and time of 16-24 hours.

[0013] In some embodiments, before removing cells from the sterilized adipose tissue using a supercritical fluid method with carbon dioxide as a solvent, the method further includes: a pretreatment step and a sterilization step, wherein the pretreatment step includes removing impurity tissue in the adipose tissue and washing it for later use; and wherein the sterilization step includes mixing and oscillating the disinfectant with the pretreated adipose tissue for sterilization, wherein the mass volume ratio of the pretreated adipose tissue to the disinfectant is 1:(10-20).

[0014] In some embodiments, the disinfection step comprises mixing the disinfectant with the pretreated adipose tissue and shaking the mixture at 25-30° C. and 150-200 rpm for 4-8 hours.

[0015] In some embodiments, the disinfectant is an aqueous solution comprising 0.02-2 v / v% peracetic acid and 4-8 v / v% ethanol, or the disinfectant is an aqueous solution comprising 0.02-2 v / v% peracetic acid and 1-5 v / v% hydrogen peroxide.

[0016] In some embodiments, the disinfectant is an aqueous solution comprising 0.02-1 v / v% peracetic acid and 4-5 v / v% ethanol.

[0017] In some embodiments, the disinfectant is an aqueous solution comprising 0.2 v / v% peracetic acid and 4 v / v% ethanol.

[0018] In some embodiments, the injectable decellularized adipose matrix preparation comprises 50-750 mmol / L sodium chloride, 0.1-10 wt % trehalose and 10-300 mmol / L pH adjuster, wherein the pH adjuster is selected from one or more of the following: Tris, phosphate, carbonate and citrate.

[0019] In some embodiments, the pH of the injectable acellular adipose matrix preparation is 5.0-9.0.

[0020] In some embodiments, the injectable decellularized adipose matrix preparation comprises 50-250 mmol / L of sodium chloride.

[0021] In some embodiments, the injectable decellularized adipose matrix preparation comprises 50-200 mmol / L of sodium chloride.

[0022] In some embodiments, the injectable decellularized adipose matrix preparation comprises 0.1-5 wt % trehalose.

[0023] In some embodiments, the injectable decellularized adipose matrix preparation comprises 1-5 wt % trehalose.

[0024] In some embodiments, the injectable decellularized adipose matrix preparation comprises 10-100 mmol / L of a pH adjuster, and the pH adjuster is Tris.

[0025] In some embodiments, the injectable decellularized adipose matrix preparation comprises 10-50 mmol / L of a pH adjuster, and the pH adjuster is Tris.

[0026] In some embodiments, the injectable decellularized adipose matrix preparation comprises 20-50 mmol / L of a pH adjuster, and the pH adjuster is Tris.

[0027] In some embodiments, the injectable decellularized adipose matrix preparation comprises 10-300 mmol / L of a pH adjuster, and the pH adjuster is phosphate, carbonate or citrate.

[0028] In some embodiments, the injectable decellularized adipose matrix preparation comprises 10-100 mmol / L of a pH adjuster, and the pH adjuster is phosphate, carbonate or citrate.

[0029] In some embodiments, the injectable decellularized adipose matrix preparation comprises 30-80 mmol / L of a pH adjuster, and the pH adjuster is phosphate, carbonate or citrate.

[0030] In some embodiments, the adipose tissue is derived from a mammal, preferably a human, sheep, cow or pig.

[0031] A second aspect of the present invention provides an injectable acellular fat matrix preparation, which is prepared according to the method described in any embodiment of the first aspect.

[0032] In some embodiments, the injectable decellularized adipose matrix preparation comprises one or more of collagen, laminin, and glycosaminoglycan; preferably, based on the total mass of the included proteins, the injectable decellularized adipose matrix preparation comprises 30%-50% collagen and 1%-5% laminin.

[0033] The third aspect of the present invention provides a method for preparing any one of the first aspect or a use of the injectable acellular fat matrix preparation of any one of the second aspect in preparing one or more of the following:

[0034] Drugs for treating or preventing osteoarthritis, medical devices for treating or preventing osteoarthritis, biomaterials for treating or preventing osteoarthritis, diagnostic reagents for diagnosing osteoarthritis, and human soft tissue fillers.

[0035] Compared with the related art, the embodiments of the present invention achieve at least the following beneficial effects:

[0036] Decellularization and defatting of adipose tissue to prepare matrix materials by physical methods is conducive to maintaining the physical and chemical properties of matrix biomaterials. Among many biomaterials, decellularized matrix, due to its unique biological properties, can not only provide good physical structural support, but also participate in regulating cell adhesion, migration, proliferation and differentiation. Compared with materials such as animal-derived or synthetic collagen, it has lower immunogenicity and better biocompatibility. It has broad application prospects in the fields of human tissue regeneration and repair, promoting vascular differentiation, inducing adipocyte production and nerve repair. It can be used for human facial soft tissue filling, joint cavity injection, uterine cavity adhesion repair, and promoting wound healing.

[0037] In addition, supercritical carbon dioxide technology is used in the preparation process of decellularized fat matrix. By combining the treatment process of pretreatment and disinfection steps, a good oil extraction effect can be achieved without using a co-extractant / carrier. The pretreatment removes the fascia tissue in the adipose tissue and reduces the surface tension of the adipose tissue. In addition, after the fascia is removed, the adipose tissue becomes granular and easier to fully contact with liquid carbon dioxide. The disinfection step is carried out in a hypotonic solution, and the hydrophilic and lipophilic characteristics of ethanol make it easier to penetrate the cell membrane of fat cells, destroy the integrity of the cell membrane in a hypotonic environment, and are more conducive to the subsequent carbon dioxide extraction effect. The yield of the matrix through this process is 4-5 times that of direct extraction with conventional organic solvents. In the final matrix, the nucleic acid content is less than 5ng / mg, and the fatty acid content is less than 50μg / mg. Furthermore, the embodiment of the present invention dissolves the decellularized fat matrix powder prepared by a specific process through a specific preparation formula to obtain an injectable injectable decellularized fat matrix preparation, so that the preparation can be stably stored at high and low temperatures.

[0038] The entire preparation process provided by the invention is simple to operate, has a high yield, and is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0040] Figure 1 This is a schematic diagram of the preparation process of the injectable acellular fat matrix preparation in Example 1;

[0041] Figure 2A The scanning electron microscopy result of the injectable acellular fat matrix preparation prepared in Experimental Group 1 in Example 1;

[0042] Figure 2B HE staining results of the injectable acellular fat matrix preparation prepared in Experimental Group 1 in Example 1;

[0043] Figure 3A The results of the Vonfrey pain threshold test of the injectable acellular fat matrix preparation prepared in Experimental Group 1 in Example 1 in treating arthritis rats;

[0044] Figure 3B The results of pathological staining of the injectable acellular fat matrix preparation prepared in Experimental Group 1 in Example 1 in the treatment of arthritis rats.

[0045] In order to facilitate the understanding of the present invention, the implementation process of the present invention is further described below in conjunction with specific implementation examples. These descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the invention. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art and are not intended to limit the present invention in any way.

[0047] In the present invention, the term "comprising" is an open expression, that is, including the contents specified in the present invention, but not excluding other contents.

[0048] The composition according to the present invention is administered in a pharmaceutically effective amount. "Pharmaceutically effective amount" as used herein refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose can be determined by parameters including the type and severity of the patient's disease, drug activity, sensitivity to the drug, administration time, route of administration and excretion rate, duration of treatment and concurrently used drugs, and other parameters well known in the medical field.

[0049] In the present invention, "prevention" refers to all actions involving the inhibition or delay of the onset of an expected disease, "treatment" refers to all actions involving the improvement or beneficial change of a disease and its metabolic abnormalities by administering a composition according to the present invention, and "alleviation" refers to all actions involving the reduction of parameters associated with an expected disease (e.g., the severity of symptoms) by administering a composition according to the present invention.

[0050] Throughout the specification, when a part "includes" / "comprises" a component, it means that it may also include other components rather than excluding components unless otherwise expressly stated. The terms "about" or "substantially" used herein are used at or near numerical values ​​when presenting allowable manufacturing and material tolerances (which are inherent in the meaning). These terms are used to prevent unscrupulous infringers from improper use of the disclosure, where correct or absolute values ​​are cited to help understand the invention.

[0051] In a first aspect, an embodiment of the present invention provides a method for preparing an injectable decellularized fat matrix preparation, comprising: removing cells from sterilized fat tissue using a supercritical fluid method with carbon dioxide as a solvent to obtain a decellularized fat matrix; freezing and grinding the decellularized fat matrix using liquid nitrogen to obtain a particle size D 90 Less than 500 μm (e.g. D 90 The injectable decellularized fat matrix preparation is prepared by dissolving the decellularized fat matrix powder in a preparation solution, filling the solution and sterilizing the solution.

[0052] In some embodiments, the freezing and grinding of the decellularized fat matrix using liquid nitrogen comprises: grinding the decellularized fat matrix in liquid nitrogen at a rotation speed of 5000rpm-8000rpm (e.g., 5500rpm, 6000rpm, 6500rpm, 7000rpm, 7500rpm), grinding 2-5 times (e.g., 2 times, 3 times, 4 times, 5 times) in total, and each grinding time is 2-5 minutes (e.g., 2 minutes, 3 minutes, 4 minutes, 5 minutes).

[0053] It can be understood that liquid nitrogen grinding includes: immersing the decellularized fat matrix in liquid nitrogen by flowing liquid nitrogen, maintaining the liquid nitrogen level at 2-3 times the volume of the decellularized fat matrix, grinding at a speed of 5000-8000 rpm, each grinding time of 2-5 minutes, and grinding 2-5 times.

[0054] In some embodiments, the step of dissolving the decellularized adipose matrix powder in a preparation solution and then filling and sterilizing the solution comprises: dissolving the decellularized adipose matrix powder in a preparation solution and then filling the solution into a syringe and sterilizing the syringe.

[0055] In some embodiments, the injectable decellularized adipose matrix preparation comprises 1 to 30 mg / ml (e.g., 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml), preferably 10 to 30 mg / ml (approximately 1-3% of the total mass of the injectable decellularized adipose matrix preparation) of the decellularized matrix powder.

[0056] In the embodiment of the present invention, the injectable acellular fat matrix preparation within the above concentration range has better stability and can maintain structural and functional stability in a high temperature and high humidity environment (eg, temperature 40°C ± 2°C, humidity 75% ± 5%).

[0057] In some embodiments, the method of sterilization is irradiation sterilization or ethylene oxide sterilization.

[0058] In some embodiments, the sterilization method is irradiation sterilization with an irradiation dose of 5-40 kGy (eg, 10 kGy, 15 kGy, 20 kGy, 25 kGy, 30 kGy, 35 kGy), preferably 20-30 kGy.

[0059] In some embodiments, the conditions of the supercritical fluid method include: pressure of 20-25Mpa (e.g., 21Mpa, 22Mpa, 23Mpa, 24Mpa, 25Mpa), temperature of 33-37°C (e.g., 34°C, 35°C, 36°C), carbon dioxide flow rate of 0.09-0.1L / min and time of 16-24 hours.

[0060] In some embodiments, before removing cells from the sterilized adipose tissue using a supercritical fluid method with carbon dioxide as a solvent, the method further includes: a pretreatment step and a sterilization step, wherein the pretreatment step includes removing impurity tissue in the adipose tissue and washing it for later use; and wherein the sterilization step includes mixing and oscillating the disinfectant with the pretreated adipose tissue for sterilization, wherein the mass volume ratio of the pretreated adipose tissue to the disinfectant is 1:(10-20).

[0061] In some embodiments, the disinfection step comprises mixing the disinfectant with the pretreated adipose tissue and shaking the mixture at 25-30° C. and 150-200 rpm for 4-8 hours.

[0062] In some embodiments, the disinfectant is an aqueous solution comprising 0.02-2 v / v% (e.g., 0.04 v / v%, 0.06 v / v%, 0.08 v / v%, 0.1 v / v%, 0.2 v / v%, 0.3 v / v%, 0.4 v / v%, 0.5 v / v%, 0.6 v / v%, 0.7 v / v%, 0.8 v / v%, 0.9 v / v%, 1 v / v%, 1.5 v / v%, 2 v / v%) of peracetic acid and 4-8 v / v% (e.g., 5 v / v%, 6 v / v%, 7 v / v%) of ethanol, or the disinfectant is an aqueous solution comprising 0.02-2 v / v% peracetic acid and 1-5 v / v% hydrogen peroxide.

[0063] In some embodiments, the disinfectant is an aqueous solution comprising 0.02-1 v / v% (e.g., 0.04 v / v%, 0.06 v / v%, 0.08 v / v%, 0.1 v / v%, 0.2 v / v%, 0.3 v / v%, 0.4 v / v%, 0.5 v / v%, 0.6 v / v%, 0.7 v / v%, 0.8 v / v%, 0.9 v / v%, 1 v / v%) peracetic acid and 4-5 v / v% ethanol, or the disinfectant is an aqueous solution comprising 0.2 v / v% peracetic acid and 4 v / v% ethanol.

[0064] In some embodiments, the injectable decellularized adipose matrix preparation comprises 50-750 mmol / L of sodium chloride (e.g., 100 mmol / L, 150 mmol / L, 200 mmol / L, 300 mmol / L, 400 mmol / L, 500 mmol / L, 600 mmol / L, 700 mmol / L), 0.1-10 wt% (e.g., 0.5 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%) of trehalose and 10-300 mmol / L (50 mmol / L, 100 mmol / L, 150 mmol / L, 200 mmol / L, 250 mmol / L) of a pH adjuster, wherein the pH adjuster is selected from one or more of the following: Tris, phosphate, carbonate and citrate.

[0065] In some embodiments, the injectable decellularized adipose matrix preparation has a pH of 5.0-9.0 (eg, pH 6.0, pH 6.5, pH 7.0, pH 7.5, pH 8.0, pH 8.5).

[0066] In some embodiments, the injectable decellularized adipose matrix preparation comprises 50-250 mmol / L of sodium chloride.

[0067] In some embodiments, the injectable decellularized adipose matrix preparation comprises 50-200 mmol / L of sodium chloride.

[0068] In some embodiments, the injectable decellularized adipose matrix preparation comprises 0.1-5 wt % trehalose.

[0069] In some embodiments, the injectable decellularized adipose matrix preparation comprises 1-5 wt % trehalose.

[0070] In some embodiments, the injectable decellularized adipose matrix preparation comprises 10-100 mmol / L of a pH adjuster, and the pH adjuster is Tris.

[0071] In some embodiments, the injectable decellularized adipose matrix preparation comprises 10-50 mmol / L of a pH adjuster, and the pH adjuster is Tris.

[0072] In some embodiments, the injectable decellularized adipose matrix preparation comprises 20-50 mmol / L of a pH adjuster, and the pH adjuster is Tris.

[0073] In some embodiments, the injectable decellularized adipose matrix preparation comprises 10-300 mmol / L of a pH adjuster, and the pH adjuster is a phosphate (e.g., sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system), a carbonate (e.g., sodium dihydrogen carbonate-disodium bicarbonate buffer system) or a citrate (e.g., citric acid-sodium citrate buffer system).

[0074] In some embodiments, the injectable decellularized adipose matrix preparation comprises 10-100 mmol / L of a pH adjuster, and the pH adjuster is phosphate, carbonate or citrate.

[0075] In some embodiments, the injectable decellularized adipose matrix preparation comprises 30-80 mmol / L of a pH adjuster, and the pH adjuster is phosphate, carbonate or citrate.

[0076] In some embodiments, the adipose tissue is derived from a mammal, preferably a human, sheep, cow or pig.

[0077] A second aspect of the present invention provides an injectable acellular fat matrix preparation, which is prepared according to the method described in any embodiment of the first aspect.

[0078] In some embodiments, the injectable decellularized adipose matrix preparation comprises one or more of collagen, laminin, and glycosaminoglycans; preferably, based on the total mass of the protein contained, the injectable decellularized adipose matrix preparation comprises 30%-50% (e.g., 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%) of collagen and 1-5% (e.g., 2%, 2.5%, 3%, 2.5%, 4%, 4.5%) of laminin.

[0079] The third aspect of the present invention provides a method for preparing any one of the first aspect or a use of the injectable acellular fat matrix preparation of any one of the second aspect in preparing one or more of the following:

[0080] Drugs for treating or preventing osteoarthritis, medical devices for treating or preventing osteoarthritis, biomaterials for treating or preventing osteoarthritis, diagnostic reagents for diagnosing osteoarthritis, and human soft tissue fillers.

[0081] The following examples are used to further illustrate the advantages and characteristics of the present method, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0082] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0083] Unless otherwise specified, the quantitative analysis experiments in the following examples were performed three times and the results were averaged.

[0084] Example 1

[0085] This embodiment provides a method for decellularizing adipose tissue. The fat comes from fat obtained by suction or surgical resection (from Shanghai Ninth People's Hospital, donated by liposuction patients, and with the patient's informed consent). The overall process is as follows: Figure 1 As shown, the following steps 1.1-1.8 are specifically included. According to steps 1.1-1.8, experimental groups 1-6 and control groups 1-3 are prepared, and the specific parameters of each group are shown in the following Table 1.

[0086] 1.1 Fat tissue cleaning: Collect fresh fat tissue samples, let them stand for 20 minutes to separate the layers, remove the blood in the fat, and stir and clean the fat tissue with cleaning solution. Repeat this process 5 times to obtain clean fat tissue.

[0087] 1.2 Pretreatment of adipose tissue: The clean adipose tissue obtained in step 1.1 is subjected to fascia and vascular tissue removal, leaving only adipose granule tissue.

[0088] 1.3 Disinfection of adipose tissue: Mix the treated adipose tissue and disinfectant at a material-liquid ratio of 1:10-1:20, and shake the shaker at a temperature of 25-30°C and a speed of 150-200 rpm for 4-8 hours;

[0089] 1.4 Degreasing of adipose tissue: Place the sterilized fat in a reactor, pump gaseous carbon dioxide into the reactor under high pressure, and prepare supercritical fluid by pressurization; maintain the pressure at 20-25Mpa, the temperature at 33-37°C, and the carbon dioxide flow rate at 0.09-0.1L / min for 16-24h, then reduce the pressure of the reactor to normal pressure, take out the extracted adipose tissue and carry out the next step of grinding.

[0090] 1.5 Liquid nitrogen cryo-grinding: Freeze the product obtained in step 1.4 with liquid nitrogen, grind the frozen matrix using an ultra-low temperature liquid nitrogen cryo-grinder, and then sieve the decellularized fat matrix powder with a particle size of less than 500 μm through a 35-mesh sieve.

[0091] 1.6 Dissolution: The decellularized matrix powder obtained in step 1.5 is dissolved in a buffer solution, which contains: 1-3% by mass of decellularized fat matrix (or 10-30 mg / mL of decellularized fat matrix, wherein 1% by mass is approximately equal to 10 mg / mL), 50-750 mmol / L of sodium chloride, 0.1-10 wt% of trehalose, and 10-300 mmol / L of a pH adjuster (10-100 mmol / L of Tris, or 10-300 mmol / L of phosphate (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system), carbonate or citrate (citric acid-sodium citrate buffer system)).

[0092] 1.7 Prefilling: Fill the solution prepared in step 1.6 into a prefilled syringe with a filling specification of 0.5-3 mL.

[0093] 1.8 Sterilization: The sterilization method is radiation sterilization or ethylene oxide sterilization; preferably, the radiation dose is 5kGy to 40kGy; more preferably, the sterilization method is to use a radiation dose of 20kGy to 30kGy.

[0094] Table 1

[0095]

[0096]

[0097] The yield of the decellularized fat matrix powder (the mass of the decellularized fat matrix powder in step 1.5) prepared by the experimental groups 1-6 and the control groups 1-3 in Table 1 above and the stability test of the decellularized matrix preparations (temperature 40°C ± 2°C, humidity 75% ± 5% for high temperature accelerated stability test, and the particle size of the decellularized fat matrix particles in each group was tested after 7 days) are shown in Table 2 below.

[0098] Table 2

[0099]

[0100] In the present embodiment, the fat matrix of experimental groups 1-6 was directly ground under liquid nitrogen conditions and degreased and decellularized by supercritical extraction, and dissolved in a specific preparation solution to obtain an injectable decellularized fat matrix preparation. As shown in Table 2 above, compared with the control group 1-3, the injectable decellularized fat matrix preparation prepared by experimental groups 1-6 had a higher yield and better stability. In addition, in the preparations prepared by experimental groups 1-6 of the present embodiment, no collagen cross-linking occurred.

[0101] Example 2

[0102] 2.1 Detection of nucleic acid residues in acellular adipose matrix preparations

[0103] The injectable acellular fat matrix preparations prepared in the experimental groups 1-6 and the control groups 1-3 were used to detect nucleic acid residues using fluorescent quantitative PCR according to the host DNA residue detection method in the Chinese Pharmacopoeia.

[0104] Fluorescence quantitative PCR instrument was purchased from ABI, USA; micro-volume UV-visible spectrophotometer NanoDrop was purchased from Thermo Fisher, USA; Varioskan Flash multifunctional microplate reader was purchased from Thermo Fisher, USA; host residual DNA extraction kit DNAExtractor kit was purchased from Thermo Fisher, USA; Power SYBRGreen MasterMix was purchased from Thermo Fisher, USA; PrepSEQ TM Residual DNA Sample Preparation Kit.

[0105] Extraction of residual human DNA in injectable decellularized adipose matrix preparations: Accurately measure 1 ml of 10 mg / ml decellularized matrix preparation, dilute it with TE solution to a final concentration of 1.5 mg / ml, and extract residual DNA according to the instructions of the DNA extractor kit.

[0106] DNA residual detection: According to the instructions of the residual DNA detection kit, the residual DNA nucleic acid content of each group of injectable acellular fat matrix preparations was measured.

[0107] The results of DNA residue detection in injectable acellular fat matrix preparations are shown in Table 3.

[0108] Table 3

[0109] Group Nucleic acid residue test results (ng / mg) Experimental Group 1 2.9 Experimental Group 2 2.17 Experimental Group 3 1.91 Experimental Group 4 1.86 Experimental Group 5 3 Experimental Group 6 2.73 Control group 1 1.87 Control group 2 75.3 Control group 3 2.4

[0110] 2.2 Characterization and detection of protein composition of decellularized fat matrix preparations

[0111] Take an appropriate amount of the adipose decellularized matrix preparation prepared in Experimental Group 1 in Example 1, use 0.5 mol / L NaOH solution to lyse the sample for 4 hours, adjust the pH to 8 with hydrochloric acid solution, add trypsin for 16 hours, and completely digest the adipose decellularized matrix sample. Then, use protein non-label quantitative technology (label free LC MS / MS) to accurately identify and relatively quantitatively analyze the proteins in the supernatant of the digested sample. Then, Gene Ontology (GO) analysis was performed on the identified proteins to determine the specific tissue cell localization classification of each protein, and the relative abundance ratio corresponding to each protein and each classification was counted.

[0112] Results: According to the data analysis of proteomics software, a total of 572 proteins were identified, 40 of which were higher than 0.3% (see Table 4 below), 14 collagens, and the total content was 32.3%. Among them, type I collagen accounted for 11.99%, which was the main protein type, followed by vimentin, etc.

[0113] Table 4

[0114]

[0115] 2.3 Electron microscopy and HE staining of decellularized adipose tissue matrix

[0116] The decellularized matrix preparation of Experimental Group 1 in Example 1 was subjected to scanning electron microscopy and HE staining. The test results are as follows: Figure 2A and Figure 2B shown.

[0117] from Figure 2A The electron microscopic results show that the decellularized matrix preparation of Experimental Group 1 in Example 1 retains the spatial three-dimensional structure of the extracellular matrix, and the matrix pore size is 20-70 μm. Figure 2B The HE staining results showed no visible cell structure, indicating that the decellularized matrix prepared by the method of Example 1 can achieve a complete decellularization effect.

[0118] Example 3 Comparison of the effects of different preparation types on the final stability of the decellularized matrix

[0119] The decellularized fat matrix powder prepared in step 1.5 of experimental group 1 in Example 1 was dissolved in physiological saline and preparation solution at 1% by weight to obtain control group 4 and experimental group 7 (containing 1% decellularized fat matrix powder (equal to 10 mg / ml decellularized fat matrix powder), 100 mmol / L sodium chloride, 5% trehalose, 50 mmol / L phosphate, pH 7.0), which were placed in a 2-8°C chromatography cabinet for long-term stability sampling. After sampling for 30 days, the particle size of the matrix solution was detected. The particle size in control group 4 was significantly larger than that in the initial D 90 Value 404.7μm, to particle size D 90 The particle size of the experimental group 7 did not change significantly after 30 days of storage, indicating that this preparation composition can significantly prolong the stability of the adipose decellularized matrix.

[0120] Table 5

[0121]

[0122] Example 4 Therapeutic effect of acellular adipose matrix preparation on osteoarthritis in rats

[0123] The SPF SD rats that passed the quarantine were selected, and the arthritis model was prepared by the sodium iodoacetate intra-articular injection method. The small animal anesthesia machine was used for isoflurane anesthesia (single: 3% for induction anesthesia, 1.5% for maintenance anesthesia; 3 rats were maintained anesthetized at the same time: a shunt device was used, and the maintenance anesthesia dose was 4-5%). After the animals were anesthetized, the rats were disinfected with 75% alcohol cotton balls around the knee joint of the left hind limb, and a single injection of 6% sodium iodoacetate (3 mg, 50 μL) was injected through the joint cavity with an insulin syringe needle. The sodium iodoacetate was filtered and sterilized with a 0.22m sterile filter membrane before injection. The normal group rats were injected with the same volume of 0.9% sodium chloride injection. After modeling, the rats were returned to the breeding cage, the bedding was changed, and they were free to eat and drink water.

[0124] After 7 days of modeling, the blank control group (normal group) and the model control group were not intervened (model control group), and the decellularized fat matrix group was administered intra-articularly once a week (divided into a low-dose group of 37.5 μg / rat / time, a medium-dose group of 75 μg / rat / time, and a high-dose group of 150 μg / rat / time, wherein the injection volume of each group was the same, and the preparation used was: the decellularized fat matrix preparation was prepared according to the method of experimental group 1, and the only difference was that the content of the decellularized fat matrix powder in the preparation was adjusted to 1 mg / mL, 2 mg / mL, and 4 mg / mL in step 1.6, so that each dose group had different decellularized fat matrix contents under the same injection volume (37.5 μL) to adapt to animal experiments, wherein the sodium chloride content was 100 mmol / L, the trehalose content was 5%, the phosphate content was 50 mmol / L, and the pH was 7.0), for a total of 4 weeks. The rat Vonfrey pain threshold was measured before and after the intervention, and the Von Frey-induced paw withdrawal reaction was measured by the Up-Down method. One week after the last intervention, tissue specimens of the femoral condyle and tibial plateau of the articular cartilage were collected, fixed with 4% paraformaldehyde solution and completely decalcified with 10% EDTA solution, and then routinely sampled, dehydrated, paraffin-embedded, and sliced ​​to 3 μm. HE staining was performed to detect the morphology of chondrocytes, safranin-fast green staining was performed to detect proteoglycan deposition, and collagen immunohistochemical staining was performed to observe the collagen content.

[0125] like Figure 3A The results showed that the pain threshold of rats in the decellularized fat matrix treatment group was significantly higher than that in the model control group, indicating that the decellularized fat matrix can alleviate the pain caused by arthritis, reduce the stimulation of pain receptors around the joints, and reduce the sensitivity of rats to pain, thus manifesting as an increase in the pain threshold.

[0126] Final pathological staining Figure 3BAs shown, for cartilage tissue, safranin can combine with acidic glycosaminoglycans in the cartilage matrix, making the cartilage tissue present a characteristic red color. The joints of rats in the model control group did not show characteristic red color, indicating that the cartilage tissue was severely damaged. After treatment with acellular fat matrix, the joint tissues of rats showed characteristic red color, indicating that it had a significant therapeutic effect. In addition, from the results of HE staining and collagen immunohistochemical staining, it can be seen that the rats in the acellular fat matrix group can reduce the infiltration of inflammatory cells in chondrocytes and increase the expression of collagen, thereby achieving the effect of treating arthritis.

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure, rather than to limit them. Although the present disclosure has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present disclosure.

[0128] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0129] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for preparing an injectable acellular fat matrix preparation, characterized in that: include: Using carbon dioxide as solvent, the cells are removed from the sterilized adipose tissue using a supercritical fluid method to obtain a decellularized adipose matrix; The decellularized fat matrix was frozen and ground using liquid nitrogen to obtain a particle size of D 90 Decellularized fat matrix powder less than 500 μm; The decellularized fat matrix powder is dissolved in a preparation solution, filled and sterilized to obtain the injectable decellularized fat matrix preparation.

2. The preparation method according to claim 1, characterized in that: The step of freezing and grinding the decellularized fat matrix using liquid nitrogen comprises: grinding the decellularized fat matrix in liquid nitrogen at a speed of 5000 rpm-8000 rpm, grinding for 2-5 times in total, and each grinding time is 2-5 minutes; Optionally, the step of dissolving the decellularized fat matrix powder in a preparation solution and then filling and sterilizing the solution comprises: dissolving the decellularized fat matrix powder in a preparation solution and then filling the solution into a syringe and sterilizing the syringe; Optionally, the injectable decellularized adipose matrix preparation comprises 1-30 mg / ml, preferably 10-30 mg / ml, of the decellularized matrix powder; Optionally, the method of sterilization is irradiation sterilization or ethylene oxide sterilization; Optionally, the sterilization method is radiation sterilization at a radiation dose of 5-40 kGy, preferably 20-30 kGy.

3. The preparation method according to claim 1 or 2, characterized in that: The conditions of the supercritical fluid method include: pressure of 20-25 MPa, temperature of 33-37° C., carbon dioxide flow rate of 0.09-0.1 L / min and time of 16-24 hours.

4. The preparation method according to any one of claims 1 to 3, characterized in that Before removing cells from the sterilized fat tissue using a supercritical fluid method with carbon dioxide as a solvent, the method further includes: a pretreatment step and a sterilization step, The pretreatment step includes removing foreign tissue from the fat tissue and washing it for later use; and The disinfection step comprises mixing and oscillating the disinfectant with the pretreated adipose tissue for disinfection, wherein the mass volume ratio of the pretreated adipose tissue to the disinfectant is 1:(10-20); Preferably, the disinfection step comprises mixing the disinfectant with the pretreated adipose tissue and shaking the mixture at 25-30° C. and 150-200 rpm for 4-8 hours.

5. The preparation method according to claim 4, characterized in that: The disinfectant is an aqueous solution comprising 0.02-2 v / v% peracetic acid and 4-8 v / v% ethanol, or the disinfectant is an aqueous solution comprising 0.02-2 v / v% peracetic acid and 1-5 v / v% hydrogen peroxide; Preferably, the disinfectant is an aqueous solution comprising 0.02-1 v / v% peracetic acid and 4-5 v / v% ethanol; More preferably, the disinfectant is an aqueous solution comprising 0.2 v / v% peracetic acid and 4 v / v% ethanol.

6. The preparation method according to any one of claims 1 to 5, characterized in that The injectable decellularized fat matrix preparation comprises 50-750 mmol / L sodium chloride, 0.1-10 wt% trehalose and 10-300 mmol / L pH adjuster, wherein the pH adjuster is selected from one or more of the following: tris (hydroxymethylaminomethane), phosphate, carbonate and citrate.

7. The preparation method according to claim 6, characterized in that: The pH of the injectable acellular fat matrix preparation is 5.0-9.0; Optionally, the injectable acellular adipose matrix preparation contains 50-250 mmol / L of sodium chloride; Optionally, the injectable acellular adipose matrix preparation contains 50-200 mmol / L of sodium chloride; Optionally, the injectable acellular adipose matrix preparation comprises 0.1-5 wt % trehalose; Optionally, the injectable acellular adipose matrix preparation comprises 1-5 wt % trehalose; Optionally, the injectable decellularized adipose matrix preparation comprises 10-100 mmol / L of a pH adjuster, and the pH adjuster is Tris; Optionally, the injectable decellularized adipose matrix preparation comprises 10-50 mmol / L of a pH adjuster, and the pH adjuster is Tris; Optionally, the injectable decellularized adipose matrix preparation comprises 20-50 mmol / L of a pH adjuster, and the pH adjuster is Tris; Optionally, the injectable decellularized adipose matrix preparation comprises 10-300 mmol / L of a pH adjuster, and the pH adjuster is phosphate, carbonate or citrate; Optionally, the injectable decellularized adipose matrix preparation comprises 10-100 mmol / L of a pH adjuster, and the pH adjuster is phosphate, carbonate or citrate; Optionally, the injectable decellularized adipose matrix preparation comprises 30-80 mmol / L of a pH adjuster, and the pH adjuster is phosphate, carbonate or citrate; Optionally, the adipose tissue is derived from mammals, preferably humans, sheep, cattle or pigs.

8. An injectable acellular fat matrix preparation, characterized in that: The injectable acellular fat matrix The preparation is prepared according to the method according to any one of claims 1-7.

9. The injectable acellular fat matrix preparation according to claim 8, characterized in that: Contains one or more of collagen, laminin, and glycosaminoglycan; preferably, based on the total mass of the included proteins, the injectable decellularized fat matrix preparation contains 30%-50% collagen and 1%-5% laminin.

10. Use of the preparation method according to any one of claims 1 to 7 or the injectable acellular fat matrix preparation according to claim 8 or 9 in the preparation of one or more of the following: Drugs for treating or preventing osteoarthritis, medical devices for treating or preventing osteoarthritis, biomaterials for treating or preventing osteoarthritis, diagnostic reagents for diagnosing osteoarthritis, and human soft tissue fillers.

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