CN111617316A - Preparation and application methods of injectable fat extracellular matrix microparticles and hydrogel prepared by pure physical method - Google Patents

Preparation and application methods of injectable fat extracellular matrix microparticles and hydrogel prepared by pure physical method Download PDF

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CN111617316A
CN111617316A CN201910146469.9A CN201910146469A CN111617316A CN 111617316 A CN111617316 A CN 111617316A CN 201910146469 A CN201910146469 A CN 201910146469A CN 111617316 A CN111617316 A CN 111617316A
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fat
extracellular matrix
layer
water
carbon dioxide
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易成刚
杨剂忠
仇利红
苏映军
熊绍恒
张兆祥
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3633—Extracellular matrix [ECM]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction

Abstract

The invention discloses a method for preparing injectable fat extracellular matrix particles and hydrogel by a pure physical method, and a preparation method and a use method thereof, wherein repeated freeze thawing, emulsification treatment, high-speed centrifugation operation and supercritical carbon dioxide (SC-CO) are adopted2) Preparing injectable fat extracellular matrix particles by a pure physical method such as an extraction method, low-temperature freeze-drying and the like; the invention not only effectively shortens the time required by treatment, improves the efficiency, but also eliminates the application of any irritant chemical substance and enzymological substance destructive to tissues, and furthest reduces the damage of the preparation method to the space structure and the protein activity of the fat extracellular matrix, so that the fat extracellular matrix can be more easily used as a soft tissue volume filler and a repair material for soft tissue defects.

Description

Preparation and application methods of injectable fat extracellular matrix microparticles and hydrogel prepared by pure physical method
Technical Field
The invention relates to the field of medical bioengineering materials, in particular to a preparation method and a use method for preparing injectable fat extracellular matrix particles by applying a pure physical method.
Background
At present, minimally invasive treatment is a development trend of repair reconstruction and plastic cosmetology. And the injection filling treatment is the first choice method for treating facial fine wrinkles and systemic volume depletion. Various filling materials are applied to the fields of repair and reconstruction and beauty, wherein artificially synthesized materials such as calcium hydroxyapatite, polyacrylamide hydrogel, polylactic acid and the like have the advantages of physical mechanics, plasticity, long-acting maintenance and the like, but the development of the artificially synthesized materials is seriously restricted due to the problems of biocompatibility, safety and the like. At present, biological materials such as collagen, hyaluronic acid and the like are clinically applied more. Human collagen and hyaluronic acid have good biocompatibility, but have the problems of fast absorption, short maintenance time, difficult raw material source, high cost and the like.
The extracellular matrix removes cell components with immunogenicity in tissues, and contains various key proteins (such as collagen, fibronectin, elastin, proteoglycan and the like), so that a good microenvironment can be provided for cell and tissue regeneration, cell behaviors can be regulated, and cell proliferation and differentiation potential can be promoted, so that the tissue regeneration of an injection part can be induced, the long-term filling effect can be achieved, and the extracellular matrix has a good application prospect in the fields of cosmetic filling, plastic surgery repair and reconstruction and the like.
Compared with other tissues, the fat is the tissue type with the most content in human body, and the fat-derived matrix can better induce the differentiation of fat cells and the regeneration of fat tissues. Chinese patent CN201610967425.9 discloses an injectable acellular fat matrix particle and application thereof in an implant, the method adopts various organic solvents such as isopropanol and the like to carry out acellular and degreasing, not only is time-consuming, but also can cause subsequent organic solvent residues to influence the biological safety and biological activity of a matrix material, so that the three-dimensional structure of an extracellular matrix is damaged, and important proteins and growth factors are eliminated, so that the microenvironment required by later-stage fat cell survival is damaged, the biological activity of the material is reduced, and the tissue regeneration induced by a scaffold material is further influenced, thereby limiting the application of the scaffold material in clinic.
Disclosure of Invention
The invention provides a preparation and use method for preparing injectable fat extracellular matrix particles by a pure physical method, which adopts repeated freeze thawing, emulsification treatment, high-speed centrifugation operation and supercritical carbon dioxide (SC-CO)2The injectable fat extracellular matrix particle is prepared by degreasing and decellularizing in pure physical modes such as extraction and the like, the time required by treatment is further shortened, the efficiency is improved, the three-dimensional structure, key protein and cell factor of the extracellular matrix are reserved, the regeneration of tissues and cells is facilitated, and the possibility is provided for the clinical application of soft tissue defect repair and cosmetic filling.
In order to solve the technical problems, the invention particularly provides the following technical scheme:
a method for preparing injectable fat extracellular matrix microparticles by a pure physical method,
the method comprises the following steps:
step 1): fat collection and processing
Obtaining human-derived or swine-derived fat particles by a liposuction technique, and rinsing the obtained fat particles with deionized water (DI water) to remove blood components; homogenizing fat by a homogenizer at 8000rpm for 2 min; then adding distilled water or hypotonic saline water, mixing uniformly, and standing for 20 minutes;
step 2): repeated freeze-thaw operation
Placing the fat particles treated by adding distilled water or hypotonic saline water in a refrigerator at-80 ℃ for 2 hours, taking out the fat particles, placing the fat particles in a water bath at 37 ℃ until the fat particles are completely thawed, and repeating the step for 5 times;
step 3): mechanical emulsification treatment
Passing the fat particles obtained in the second step through a metal adapter with the inner diameter of 1.0mm by using an injector or a fat emulsion device; 60 to 120 transfers back and forth at a uniform rate of 10 ml/s;
step 4): high speed centrifugal operation
Collecting the fat particles in the third step, loading the fat particles in a centrifugal tube, and carrying out centrifugal operation by high-speed centrifugation to obtain four layers of layers, wherein the four layers of layers sequentially comprise from top to bottom:
layer 1: a fat layer, which is extracted for use in the next step five;
layer 2: a fat matrix mixing layer; detecting the fat matrix mixed layer, wherein if cell components exist, collecting the cell components, adding distilled water or hypotonic saline water, uniformly mixing, standing for 20 minutes, and repeating the operations of the step 2), the step 3) and the step 4) once or even several times until no cell residue exists; collecting adipose-derived matrix mixed tissue;
layer 3: removing the water layer;
layer 4: removing the cell layer;
step 5): high speed centrifugation degreasing
Adding the grease layer collected in the layer 1 in the step 4) into the fat-matrix mixed tissue, then carrying out the operation of the step 3), and then carrying out high-speed centrifugation to separate out most of grease on the upper layer for removal; the middle layer is a fat matrix mixed tissue and is reserved, and a small amount of water in the lower layer can be removed;
step 6): continuously degreasing
Analyzing the oil content of the fat matrix mixed tissue in the step 5), if the oil still exists, repeating the step 5) until no oil component can be detected; obtaining fat-derived extracellular matrix;
step 7): supercritical carbon dioxide extraction degreasing treatment
(1) Transferring the fat matrix mixed layer collected in the layer 2 in the step 4) into an extractor of a supercritical carbon dioxide extraction device;
(2) firstly, adjusting the pressure to 18-19 MPa through a pressure regulator; adjusting the temperature to 36.5-37.5 ℃ by a temperature regulator, wherein the temperature is CO2The flow rate of the water is adjusted to 23-25L/h; at the moment, the carbon dioxide is in a supercritical fluid state; flowing supercritical carbon dioxide fluid through an extractor and collectingThe obtained fat particles are subjected to contact reaction;
(3) the lipid and the cells are subjected to a dissolving reaction with the supercritical carbon dioxide fluid and enter the separator along with the fluid, and the amino acid substances with strong polarity and the extracellular matrix are retained in the extractor; in the separator, the temperature is adjusted to be higher than 32 ℃ and the pressure is controlled to be lower than 7MPa, so that the supercritical state of CO2 is recovered to be in a gas state, and the separation of CO2 and the extracted grease is realized;
(4) passing supercritical carbon dioxide fluid through CO2Liquefied in the liquefier and collected with CO2In the storage device, the next cycle extraction is carried out, and the cycle is repeatedly carried out for a plurality of times until the lipid and cells in the fat particles are completely extracted;
(5) the continuous cycle time of the whole process is about 3 hours; obtaining fat-derived extracellular matrix;
step 8): low temperature preservation
Cooling the fat-derived extracellular matrix obtained in the step 6) and the step 7) to-80 ℃ in a programmed manner, and sterilizing the fat-derived extracellular matrix by gamma ray irradiation (25-35 kGy) after freeze-drying; vacuum placing in sealed bag, and storing at 4 deg.C for use.
A method for preparing an adipose extracellular matrix hydrogel by using the adipose extracellular matrix microparticles of claim 1, wherein the method comprises the following steps:
the obtained fat extracellular matrix microparticles were fully dissolved with pepsin at room temperature, then the pH was adjusted to 7.4, and the mixture was placed at 37 ℃ to form a fat extracellular matrix hydrogel.
A method of using the fat extracellular matrix hydrogel of claim 2, which is topically injected through a 27g needle for facial rejuvenation and soft tissue injury repair.
Compared with the prior art, the invention has the following beneficial effects:
the supercritical preparation method of the injectable fat extracellular matrix particle eliminates the application of any irritant chemical substance and enzyme substance which is destructive to tissues, further shortens the time required by treatment, improves the efficiency, reserves the three-dimensional structure, key protein and cytokine of the extracellular matrix, and provides possibility for the clinical application of soft tissue defect repair and plastic surgery.
The injectable fat extracellular matrix particles are prepared by a pure physical method, on one hand, the dsDNA content of the extracellular matrix biomaterial obtained by the pure physical methods of repeated freeze thawing, emulsification treatment, high-speed centrifugal operation, supercritical carbon dioxide extraction and the like is detected to be lower than the threshold level of 50ng/mg, which is considered as the safety range of clinical application; on the other hand, purely physical methods retain not only key proteins and growth factors such as collagens of types I, II, IV, VI, etc., elastin (elastin), fibronectin (fibronectin), laminin (laminin), etc., but also other biological factors such as glycosaminoglycans (GAGs) and growth factors such as basic fibroblast growth factor (bFGF) and Vascular Endothelial Growth Factor (VEGF). The retention of these key proteins and growth factors is critical for the ability of the extracellular matrix (ECM) to repair and regenerate fat in vivo from soft tissues.
Drawings
FIG. 1 is a process flow diagram of the preparation method of injectable adipose extracellular matrix microparticles by the purely physical method of the present invention;
FIG. 2 is a flow chart of the process of the present invention for degreasing by supercritical carbon dioxide extraction;
FIG. 3 is C02Pressure versus temperature and volumetric mass.
Detailed Description
In order to facilitate understanding of the objects, technical solutions and effects of the present invention, the present invention will be further described in detail with reference to examples.
As shown in FIG. 1, the method for preparing injectable adipose extracellular matrix microparticles by a purely physical method according to the present invention,
the method comprises the following steps:
step 1): fat collection and processing
Obtaining human-derived or swine-derived fat particles by a liposuction technique, and rinsing the obtained fat particles with deionized water (DI water) to remove blood components; homogenizing fat by a homogenizer at 8000rpm for 2 min; then adding distilled water or hypotonic saline water, mixing uniformly, and standing for 20 minutes;
step 2): repeated freeze-thaw operation
Placing the fat particles treated by adding distilled water or hypotonic saline water in a refrigerator at-80 ℃ for 2 hours, taking out the fat particles, placing the fat particles in a water bath at 37 ℃ until the fat particles are completely thawed, and repeating the step for 5 times;
step 3): mechanical emulsification treatment
Passing the fat particles obtained in the second step through a metal adapter with the inner diameter of 1.0mm by using an injector or a fat emulsion device; 60 to 120 transfers back and forth at a uniform rate of 10 ml/s;
step 4): high speed centrifugal operation
Collecting the fat particles in the third step, loading the fat particles in a centrifugal tube, and carrying out centrifugal operation by high-speed centrifugation to obtain four layers of layers, wherein the four layers of layers sequentially comprise from top to bottom:
layer 1: a fat layer, which is extracted for use in the next step five;
layer 2: a fat matrix mixing layer; detecting the fat matrix mixed layer, wherein if cell components exist, collecting the cell components, adding distilled water or hypotonic saline water, uniformly mixing, standing for 20 minutes, and repeating the operations of the step 2), the step 3) and the step 4) once or even several times until no cell residue exists; collecting adipose-derived matrix mixed tissue;
layer 3: removing the water layer;
layer 4: removing the cell layer;
step 5): high speed centrifugation degreasing
Adding the grease layer collected in the layer 1 in the step 4) into the fat-matrix mixed tissue, then carrying out the operation of the step 3), and then carrying out high-speed centrifugation to separate out most of grease on the upper layer for removal; the middle layer is a fat matrix mixed tissue and is reserved, and a small amount of water in the lower layer can be removed;
step 6): continuously degreasing
Analyzing the oil content of the fat matrix mixed tissue in the step 5), if the oil still exists, repeating the step 5) until no oil component can be detected; obtaining fat-derived extracellular matrix;
as shown in fig. 2, step 7): supercritical carbon dioxide extraction degreasing treatment
(1) Transferring the fat matrix mixed layer collected in the layer 2 in the step 4) into an extractor of a supercritical carbon dioxide extraction device;
(2) firstly, adjusting the pressure to 18-19 MPa through a pressure regulator; adjusting the temperature to 36.5-37.5 ℃ by a temperature regulator, wherein the temperature is CO2The flow rate of the water is adjusted to 23-25L/h; at the moment, the carbon dioxide is in a supercritical fluid state; flowing supercritical carbon dioxide fluid through an extractor to perform contact reaction with the collected fat particles;
(3) the lipid and the cells are subjected to a dissolving reaction with the supercritical carbon dioxide fluid and enter the separator along with the fluid, and the amino acid substances with strong polarity and the extracellular matrix are retained in the extractor; in the separator, the temperature is adjusted to be higher than 32 ℃ and the pressure is controlled to be lower than 7MPa, so that the supercritical state of CO2 is recovered to be in a gas state, and the separation of CO2 and the extracted grease is realized;
(4) passing supercritical carbon dioxide fluid through CO2Liquefied in the liquefier and collected with CO2In the storage device, the next cycle extraction is carried out, and the cycle is repeatedly carried out for a plurality of times until the lipid and cells in the fat particles are completely extracted;
(5) the continuous cycle time of the whole process is about 3 hours; obtaining fat-derived extracellular matrix;
step 8): low temperature preservation
Cooling the fat-derived extracellular matrix obtained in the step 6) and the step 7) to-80 ℃ in a programmed manner, and sterilizing the fat-derived extracellular matrix by gamma ray irradiation (25-35 kGy) after freeze-drying; vacuum placing in sealed bag, and storing at 4 deg.C for use.
A method for preparing an adipose extracellular matrix hydrogel by using the adipose extracellular matrix microparticles of claim 1, wherein the method comprises the following steps:
the obtained fat extracellular matrix microparticles were fully dissolved with pepsin at room temperature, then the pH was adjusted to 7.4, and the mixture was placed at 37 ℃ to form a fat extracellular matrix hydrogel.
Wherein the physical crosslinking method includes using at least one of ultraviolet light, electron beam, and gamma ray radiation. The chemical crosslinking method comprises at least one of carbodiimide, glutaraldehyde and genipin as crosslinking agents.
A method of using the fat extracellular matrix hydrogel of claim 2, which is topically injected through a 27g needle for facial rejuvenation and soft tissue injury repair.
The injectable fat extracellular matrix particle is prepared by adopting a pure physical preparation method, and fat tissues are collected by a fat sucking operation, and are degreased and decellularized in pure physical modes of repeated freeze thawing, mechanical emulsification and erosization treatment, high-speed centrifugal operation, supercritical carbon dioxide extraction and the like; the physical fat cell breaking and degreasing method and the mild cell removing technology are simple and convenient to operate, the time required by treatment is effectively shortened, and the efficiency is improved. The application of irritant chemical substances and enzymological substances destructive to tissues is eliminated, the three-dimensional structure and active ingredients of the tissues are reserved, the obtained material is closer to the natural adipose tissue microenvironment of a human body, a small 27g needle can be used for injection, and the proliferation and differentiation of cells are facilitated after the injection, so that the repair and regeneration of the tissues are promoted, and the long-term filling effect is achieved. The product of the invention is suitable for facial rejuvenation, soft tissue defect repair and the like.
The injectable fat extracellular matrix particles are prepared by a pure physical method, wherein the step 7) is carried out degreasing treatment by using a supercritical carbon dioxide extraction method, and the technical principle is as follows:
pure substances can present state changes of liquid, gas, solid and the like according to different temperatures and pressures. When a certain temperature and pressure are reached, the phenomenon that the interface between liquid and gas disappears appears in the substance, and the point is called the temporary situationAnd (4) a boundary point. Near the critical point, there is a phenomenon in which the properties of all substances such as density, viscosity, solubility, heat capacity, and dielectric constant of the Fluid change rapidly, and a Fluid having a temperature and a pressure higher than the critical point is called a Supercritical Fluid (Supercritical Fluid). The critical point of carbon dioxide is not only easy to reach (31.3 deg.C, 7.15MPa), but also has the advantages of no toxicity, no smell, no flammability and explosiveness, low energy consumption, etc. CO in supercritical state2The density is greatly increased to greatly increase the solubility of solute, then the supercritical fluid is changed into common gas by means of decompression and temperature rise, and the common gas and the extracted substance are completely or basically separated, so that the purposes of separation and purification are achieved, namely supercritical CO2And (3) an extraction process.
Supercritical CO2Is very sensitive to temperature and pressure changes, and CO can be adjusted by adjusting the pressure and the temperature2And further adjusting the dissolving capacity of the target extract. The extracts obtained in the respective pressure ranges are not single, and the conditions can be controlled to obtain the mixed components in the optimum ratio.
FIG. 3 is a C0 view as shown in FIG. 32Pressure versus temperature and volumetric mass;
line A to Tp in FIG. 3CO2Sublimation curve of gas-solid equilibrium, line B-Tp representing CO2Liquid-solid equilibrium melting curve, Tp-Cp line representing CO2The vapor-liquid equilibrium vapor pressure curve of (1). Tp is a triple point where gas-liquid-solid phases coexist.
Increasing pressure and temperature along the gas-liquid saturation curve reaches the critical point Cp. When the temperature reaches above 31.06 ℃ and the pressure is above 7.39MPa, the shadow part of the system is in a CO2 supercritical state. CO22The supercritical fluid is between gas and liquid, has the dual characteristics of gas and liquid, and has the density similar to that of the liquid and the viscosity similar to that of the gas; that is, the density is hundreds of times higher than the density of gas, and the value is equivalent to that of liquid; the viscosity is two orders of magnitude smaller than that of liquid, and the value of the viscosity is equivalent to that of gas; the diffusion coefficient is between that of gas and liquid, about 1/100 for gas, and hundreds of times greater than for liquid. In the whole supercritical CO2In the process of extraction, the extraction liquid is extracted,the pressure of the extraction is one of the most important parameters. At a certain extraction temperature, the pressure is increased, and CO is added2The density of the liquid increases and its solubility increases.
However, for the extracellular matrix of adipose tissue, the higher the extraction pressure is not the better. According to the domestic and foreign studies, it has been found that when the pressure reaches 25MPa or more at a constant temperature (37 ℃), the fine structural components of the fat extracellular matrix, such as-OH, -COOH-based substances, glycosides and amino acids, collagen (I, II, IV, VI, etc.) and stromal cell proteins (cysteine-rich acidic secreted proteins, thrombospondin, osteopontin, cytoadhesin, etc.), etc., are affected; using the above CO2According to the characteristic of the curve, in the supercritical carbon dioxide extraction process, the extraction pressure is maintained at 18-19 MPa, the extraction temperature is maintained at 36.5-37.5 ℃, and the influence on the ultrastructure of the extracellular matrix as small as possible and the retention to the maximum extent can be realized while the mild extraction is realized. Introducing CO2The circulation flow of the reactor is kept at 23-25L/h, and the reactor is continuously circulated for 3 hours; at the same time, the above CO is utilized2The characteristic of the graph, the temperature in the separator is adjusted to be above 32 ℃ and the pressure is controlled to be below 7MPa, so that the CO can be obtained2The supercritical state of the gas is restored to a gas state, thereby realizing CO2And separating the extracted grease.
In addition, the temperature is also supercritical CO2Another important parameter in the extraction process. As shown in fig. 3, when the pressure is constant (18 to 19MPa), the density of the supercritical CO2 fluid decreases with the increase of the temperature, the dissolving capacity decreases with the decrease of the density, and the extraction of the amount of the grease is further affected. The invention utilizes the characteristic, when the pressure is kept within a certain range (18-19 MPa), the temperature is adjusted to be close to the normal temperature (36.5-37.5 ℃) of the human body, and the destructive power to the fat extracellular matrix is reduced to the minimum degree, and the better dissolving capacity of the supercritical CO2 fluid can be realized.
Therefore, in the whole supercritical carbon dioxide extraction process, when the pressure is 18-19 MPa and the temperature is 36.5-37.5 ℃, the three-dimensional structure and the active ingredients of the fat extracellular matrix can be well reserved, the obtained material is closer to the natural fat tissue microenvironment of a human body, and the proliferation and differentiation of fat cells in and out of the human body are facilitated, so that the tissue repair and regeneration are promoted, and the long-term filling effect is achieved.
The present invention has been further described with reference to the examples, but the present invention is not limited to the above-described embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.

Claims (3)

1. A method for preparing injectable fat extracellular matrix microparticles by a pure physical method is characterized in that,
the method comprises the following steps:
step 1): fat collection and processing
Obtaining human-derived or swine-derived fat particles by a liposuction technique, and rinsing the obtained fat particles with deionized water (DI water) to remove blood components; homogenizing fat by a homogenizer at 8000rpm for 2 min; then adding distilled water or hypotonic saline water, mixing uniformly, and standing for 20 minutes;
step 2): repeated freeze-thaw operation
Placing the fat particles treated by adding distilled water or hypotonic saline water in a refrigerator at-80 ℃ for 2 hours, taking out the fat particles, placing the fat particles in a water bath at 37 ℃ until the fat particles are completely thawed, and repeating the step for 5 times;
step 3): mechanical emulsification treatment
Passing the fat particles obtained in the second step through a metal adapter with the inner diameter of 1.0mm by using an injector or a fat emulsion device; 60 to 120 transfers back and forth at a uniform rate of 10 ml/s;
step 4): high speed centrifugal operation
Collecting the fat particles in the third step, loading the fat particles in a centrifugal tube, and carrying out centrifugal operation by high-speed centrifugation to obtain four layers of layers, wherein the four layers of layers sequentially comprise from top to bottom:
layer 1: a fat layer, which is extracted for use in the next step five;
layer 2: a fat matrix mixing layer; detecting the fat matrix mixed layer, wherein if cell components exist, collecting the cell components, adding distilled water or hypotonic saline water, uniformly mixing, standing for 20 minutes, and repeating the operations of the step 2), the step 3) and the step 4) once or even several times until no cell residue exists; collecting adipose-derived matrix mixed tissue;
layer 3: removing the water layer;
layer 4: removing the cell layer;
step 5): high speed centrifugation degreasing
Adding the grease layer collected in the layer 1 in the step 4) into the fat-matrix mixed tissue, then carrying out the operation of the step 3), and then carrying out high-speed centrifugation to separate out most of grease on the upper layer for removal; the middle layer is a fat matrix mixed tissue and is reserved, and a small amount of water in the lower layer can be removed;
step 6): continuously degreasing
Analyzing the oil content of the fat matrix mixed tissue in the step 5), if the oil still exists, repeating the step 5) until no oil component can be detected; obtaining fat-derived extracellular matrix;
step 7): supercritical carbon dioxide extraction degreasing treatment
(1) Transferring the fat matrix mixed layer collected in the layer 2 in the step 4) into an extractor of a supercritical carbon dioxide extraction device;
(2) firstly, adjusting the pressure to 18-19 MPa through a pressure regulator; adjusting the temperature to 36.5-37.5 ℃ by a temperature regulator, wherein the temperature is CO2The flow rate of the water is adjusted to 23-25L/h; at the moment, the carbon dioxide is in a supercritical fluid state; flowing supercritical carbon dioxide fluid through an extractor to perform contact reaction with the collected fat particles;
(3) the lipid and the cells are subjected to a dissolving reaction with the supercritical carbon dioxide fluid and enter the separator along with the fluid, and the amino acid substances with strong polarity and the extracellular matrix are retained in the extractor; in the separator, the temperature is adjusted to be higher than 32 ℃ and the pressure is controlled to be lower than 7MPa, so that the supercritical state of CO2 is recovered to be in a gas state, and the separation of CO2 and the extracted grease is realized;
(4) passing supercritical carbon dioxide fluid through CO2Liquefied in the liquefier and collected with CO2In the storage device, the next cycle extraction is carried out, and the cycle is repeatedly carried out for a plurality of times until the lipid and cells in the fat particles are completely extracted;
(5) the continuous cycle time of the whole process is about 3 hours; obtaining fat-derived extracellular matrix;
step 8): low temperature preservation
Cooling the fat-derived extracellular matrix obtained in the step 6) and the step 7) to-80 ℃ in a programmed manner, and sterilizing the fat-derived extracellular matrix by gamma ray irradiation (25-35 kGy) after freeze-drying; vacuum placing in sealed bag, and storing at 4 deg.C for use.
2. A method for preparing an adipose extracellular matrix hydrogel by using the adipose extracellular matrix microparticles of claim 1, wherein the method comprises the following steps:
the obtained fat extracellular matrix microparticles were fully dissolved with pepsin at room temperature, then the pH was adjusted to 7.4, and the mixture was placed at 37 ℃ to form a fat extracellular matrix hydrogel.
3. A method of using the adipose extracellular matrix hydrogel of claim 2, wherein the method comprises:
the fat extracellular matrix hydrogel can be injected locally through a 27g small needle, and can be used for facial rejuvenation and soft tissue injury repair.
CN201910146469.9A 2019-02-27 2019-02-27 Preparation and application methods of injectable fat extracellular matrix microparticles and hydrogel prepared by pure physical method Pending CN111617316A (en)

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