A porous collagen microsphere and a preparation method and application thereof

Abstract

This invention relates to porous collagen microspheres, their preparation method, and applications. The method includes: uniformly mixing collagen and amino acids with hydrochloric acid solution to obtain a collagen / amino acid mixed solution; granulating the collagen / amino acid mixed solution by liquid nitrogen spraying to obtain frozen microspheres; and subjecting the frozen microspheres to a first freeze-drying, cross-linking, and a second freeze-drying process to obtain porous collagen microspheres. The porous collagen microspheres prepared by this invention have an interconnected three-dimensional porous network structure, which can simulate the complex topology of natural ECM. The resulting microspheres efficiently retain regenerative active ingredients such as bFGF, VEGF, TGF-β1, and glycosaminoglycans, making them particularly suitable as core materials for growth factor sustained-release microspheres. They can provide a basis for achieving the time-sequential release of VEGF and PDGF-BB, which is beneficial for promoting blood circulation and improving fat survival rate.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and particularly relates to a porous collagen microsphere, its preparation method and application. Background Technology

[0002] Adipose-derived stem cells in fat act like "seeds," secreting abundant growth factors (such as VEGF, HGF, and FGF) to promote angiogenesis and the synthesis of collagen and elastin intermediates in the transplanted area. The effect is not just a "puffing up," but also thicker, firmer, and more radiant skin, achieving true "skin rejuvenation." Although there is an absorption rate, once the fat cells successfully establish blood supply, they become permanently viable autologous tissue. This is a huge attraction for those seeking long-term solutions and unwilling to make frequent trips to the hospital.

[0003] Fat grafting in the field of aesthetic medicine is one of the most popular and demanding regenerative aesthetic procedures for doctors. It has evolved from a simple "liposuction-injection" procedure into a sophisticated autologous tissue transplantation and regeneration technique. Fat cells are large, fragile, and sensitive to hypoxia, requiring rapid establishment of blood supply to survive. If the center of the injected fat mass is too far from a blood vessel (>200 micrometers), it will die due to ischemia and become a processed product. To achieve better results, researchers have developed injectable hydrogel scaffolds such as hyaluronic acid, gelatin, and decellularized fat matrix hydrogels. These scaffolds function to evenly disperse cells, prevent cell aggregation leading to necrosis, and provide immediate physical space, but they are gradually metabolized, with limited effect on establishing blood supply. Therefore, fat regeneration requires a three-dimensional microenvironment "scaffold" that can rapidly induce angiogenesis and mimic the biochemical / mechanical properties of natural fat ECM.

[0004] Natural extracellular matrix (ECM) has a three-dimensional structure and comprehensive biological activity, but it also faces the problems of complex composition and difficulty in standardization. Residual DNA or cell membrane components, α-Gal antigens, etc. may trigger immune rejection or inflammatory responses. Collagen with telopeptide removed by enzymatic cleavage or recombinant technology has significantly reduced immunogenicity and higher safety, but it lacks the multiple signaling molecules and complex topological structures of ECM, has limited ability to guide cell behavior, and its tissue regeneration effect may not be as good as that of ECM.

[0005] Against this backdrop, injectable microcarrier scaffold materials have become a research hotspot in the field of tissue engineering. Among them, collagen microspheres, as an injectable scaffold material, have good biocompatibility and biodegradability. However, collagen microspheres prepared in the prior art have one or more of the following problems: (1) low porosity and poor pore connectivity, making it difficult to meet the needs of cell ingrowth and material exchange; (2) poor mechanical strength, and the structure is prone to collapse when exposed to water; (3) lack of pore-forming technology that can precisely control the pore structure. Existing technologies mostly use organic solvent pore-forming methods (such as isooctane, camphene, toluene, etc.) or emulsion phase separation methods, but organic solvent residues may cause biosafety issues, and the pore structure is difficult to control precisely.

[0006] Therefore, developing porous collagen microspheres with controllable pore structure, good mechanical strength, and high biocompatibility has significant clinical application value. In particular, if these porous collagen microspheres can be further developed into a sustained-release system capable of loading growth factors, it is expected to provide an ideal "artificial extracellular matrix" scaffold for fat grafting, solving the problem of physical support for fat cells and promoting blood supply by inducing angiogenesis.

[0007] In summary, it is essential to provide a porous collagen microsphere, its preparation method, and its application. Summary of the Invention

[0008] To address one or more technical problems existing in the prior art, this invention provides porous collagen microspheres, their preparation method, and their applications.

[0009] The present invention provides a method for preparing porous collagen microspheres in a first aspect, the method comprising the following steps: (1) Mix collagen and amino acids evenly with hydrochloric acid solution to obtain collagen / amino acid mixed solution; (2) The collagen / amino acid mixture solution was granulated by liquid nitrogen spraying to obtain frozen microspheres; (3) The frozen microspheres were subjected to first freeze-drying, cross-linking and second freeze-drying in sequence to prepare porous collagen microspheres.

[0010] The present invention provides, in a second aspect, porous collagen microspheres prepared by the preparation method described in the first aspect of the present invention.

[0011] In a third aspect, the present invention provides the application of porous collagen microspheres prepared by the preparation method described in the first aspect of the present invention as core materials for tissue engineering scaffolds, cell culture carriers, drug delivery carriers, or core-shell structured microspheres; preferably, the core-shell structured microspheres are dual growth factor sustained-release microspheres with a core-shell structure; preferably, the dual growth factors are platelet-derived growth factor PDGF-BB and vascular endothelial growth factor VEGF.

[0012] Compared with the prior art, the present invention has at least the following beneficial effects: (1) This invention is the first to use amino acids as pore-forming agents to prepare porous collagen microspheres. As a naturally occurring substance in the body, amino acids have good biocompatibility, no risk of organic solvent residue, and good compatibility with collagen, and will not affect the bioactivity of collagen. The porous collagen microspheres prepared by the method of this invention have a three-dimensional porous network with interconnected channels, which can simulate the complex topology of natural ECM. This structure is conducive to cell ingrowth, nutrient exchange and metabolic waste excretion. At the same time, the porous structure is also conducive to the loading and slow release of growth factors: when used to load PDGF-BB, the porous structure can effectively encapsulate PDGF-BB inside the microsphere, achieving long-term slow release. The porous collagen microspheres of this invention have excellent mechanical strength. After soaking in water for 24 hours, they still maintain an intact structure without significant collapse, which can meet the mechanical requirements of syringe injection and in vivo implantation.

[0013] (2) The present invention has found that the porous collagen microspheres of the present invention are particularly suitable for use as core materials in tissue engineering scaffolds, cell culture carriers, drug delivery carriers, or core-shell structured microspheres, and more preferably as core materials in core-shell structured microspheres. Specifically, for example, core-shell structured microspheres prepared by loading PDGF-BB onto the porous collagen microspheres of the present invention as core materials and then coating them with VEGF-collagen shells using microfluidic technology can exhibit excellent time-sequential release properties, forming a time-sequential release system of "rapid release of VEGF from the shell / slow release of PDGF-BB from the core", which simulates angiogenesis. The physiological process of fat grafting (first VEGF induces budding, then PDGF-BB promotes maturation) provides an ideal artificial ECM microenvironment for fat grafting. The porous collagen microspheres of this invention are perfectly suited for preparing core-shell structured dual growth factor sustained-release microspheres. They are the core material for the latter to achieve the biochemical / mechanical properties and time-sequential release function of natural ECM. They can sustainably release vascular endothelial growth factor and platelet-derived growth factor, promote the successful establishment of blood supply in fat cells, provide an ideal artificial ECM scaffold for fat grafting, solve the problem of fat cell ischemia and necrosis, and ultimately help achieve the therapeutic effect of "skin rejuvenation".

[0014] (3) In some preferred technical solutions of the present invention, the collagen used is self-made adipose-derived decellularized collagen. Through the optimization of the gradient degreasing strategy, pepsin enzymatic hydrolysis process and pH gradient dialysis of the present invention, the key active ingredients for promoting regeneration in adipose-derived collagen are efficiently retained. Specifically, the present invention adopts a three-step progressive gradient degreasing with an increasing material-to-liquid ratio, which can thoroughly remove oil, avoid inflammatory reactions and barrier effects caused by residual oil, and effectively protect the collagen conformation and growth factor binding sites in the ECM. The pepsin enzymatic hydrolysis minimizes the degradation loss of heat-sensitive active ingredients such as bFGF, VEGF, and TGF-β1. The pH gradient dialysis effectively removes pepsin while retaining effective ingredients such as glycosaminoglycans, while avoiding collagen aggregation and precipitation and growth factor inactivation caused by direct rapid dialysis. More importantly, the above The active ingredients are efficiently preserved in the subsequent porous collagen microsphere preparation process of this invention. This means that the porous collagen microspheres of this invention not only provide a three-dimensional physical scaffold, but also carry bioactive regenerative signaling molecules: bFGF can promote fibroblast proliferation and collagen synthesis, which helps to restructure and improve the firmness of the skin in the transplantation area; VEGF can promote angiogenesis around the microspheres, improve blood supply, and provide necessary nutrients and oxygen for the survival of adipocytes; TGF-β1 participates in extracellular matrix synthesis and tissue repair, and promotes the production of collagen and elastin; glycosaminoglycans maintain the moisture in the microspheres and protect the activity of growth factors, while providing a good hydration environment for tissues. The combination of these active ingredients with the porous microspheres makes the product of this invention have the dual functions of physical scaffold and biological signaling, which is conducive to promoting the therapeutic effect of "skin rejuvenation". Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is an image showing the HE staining results of decellularized collagen in fat in Example 1 of this invention; Figure 2 This is an image of Oil Red O staining results of decellularized collagen in fat in Example 1 of the present invention; in the image, (a) and (b) are Oil Red O staining results under the same magnification but different fields of view; Figure 3 The image shows the surface morphology of the porous collagen microspheres prepared in Example 1 of this invention as observed by scanning electron microscopy (SEM); in the image, (b) is a magnified view of (a). Figure 4 The image shows the cross-sectional morphology of the porous collagen microspheres prepared in Example 1 of this invention, observed using scanning electron microscopy (SEM). Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0018] The present invention provides a method for preparing porous collagen microspheres in a first aspect, the method comprising the following steps: (1) Mix collagen and amino acids evenly with hydrochloric acid solution to obtain collagen / amino acid mixed solution; (2) The collagen / amino acid mixture solution was granulated by liquid nitrogen spraying to obtain frozen microspheres; (3) The frozen microspheres were subjected to first freeze-drying, cross-linking and second freeze-drying in sequence to prepare porous collagen microspheres.

[0019] Existing technologies CN117482292A and CN121801122A report the preparation of porous collagen microspheres using electrospinning combined with glutaraldehyde crosslinking and radiation crosslinking. These technologies both achieve control over the overall size of the microspheres, but their pore structure relies on passive pore formation, making it impossible to independently control the pore size and porosity of the internal porous structure. Furthermore, these processes depend on precision equipment, making scale-up difficult. In contrast, this invention uses amino acids as pore-forming agents. After liquid nitrogen spray granulation and freeze-drying to form pores, the porous collagen microspheres are obtained through EDC hydrochloride / NHS crosslinking. This invention uses amino acids for pore formation, allowing precise control of the pore size and porosity of the internal porous structure of the microspheres. This enables active and precise design of the internal pore structure, thereby optimizing cell adhesion and migration performance while ensuring mechanical strength. Moreover, this invention does not require high-voltage electrostatic or microfluidic equipment, resulting in a wider process window.

[0020] This invention is the first to use amino acids as pore-forming agents to prepare porous collagen microspheres. Amino acids, as naturally occurring substances in living organisms, possess excellent biocompatibility, pose no risk of organic solvent residue, and exhibit good compatibility with collagen, without affecting its bioactivity. The porous collagen microspheres prepared by this method form an interconnected three-dimensional porous network, mimicking the complex topology of the natural ECM. This structure facilitates cell ingrowth, nutrient exchange, and the removal of metabolic waste. Simultaneously, this porous structure also facilitates the loading and sustained release of growth factors: when used to load PDGF-BB, the porous structure effectively encapsulates PDGF-BB within the microspheres, achieving long-term, slow release. The porous collagen microspheres of this invention exhibit excellent mechanical strength, maintaining their intact structure without significant collapse after immersion in water for 24 hours, meeting the mechanical requirements for syringe injection and in vivo implantation.

[0021] According to some preferred embodiments, the concentration of the hydrochloric acid solution (hydrochloric acid aqueous solution) is 0.005~0.015mol / L; the concentration of collagen in the collagen / amino acid mixed solution is 1~3w / v%; in this invention, the unit "w / v%" means "g / 100mL"; the mass amount of the amino acid is 5~15% of the mass amount of the collagen, preferably 10%.

[0022] In this invention, it is preferred that the amount of amino acids added is 5% to 15% of the collagen (dry weight). This invention has found that if the amount of amino acids added is too low, the pore-forming effect is insufficient, the porosity is low, the pore connectivity is poor, and it is difficult for cells to migrate into the microspheres for growth. If the amount of amino acids added is too high, although a higher porosity can be obtained, the mechanical strength of the resulting porous collagen microspheres is significantly reduced, making it difficult to meet the mechanical requirements for injection and in vivo implantation.

[0023] According to some preferred embodiments, the amino acid is composed of glycine, proline and hydroxyproline in a mass ratio of 2:(0.5~1.5):(0.5~1.5); or the amino acid is composed of glycine, hydroxyproline and glutamine in a mass ratio of 2:(0.5~1.5):(0.5~1.5).

[0024] Preferably, the amino acids in this invention are composed of glycine, proline, and hydroxyproline in a mass ratio of 2:(0.5~1.5):(0.5~1.5), or glycine, hydroxyproline, and glutamine in a mass ratio of 2:(0.5~1.5):(0.5~1.5). This invention reveals that glycine, as a core skeletal component, influences ice crystal nucleation and growth through hydrogen bonding, forming uniform micropores. Proline and hydroxyproline, as characteristic amino acids of collagen, exhibit a synergistic effect between the hydrophilic hydroxyl group of hydroxyproline and the pyrrole ring spatial effect of proline, which can also promote the formation of interconnected channels. The side-chain amide group of glutamine is highly hydrophilic, significantly altering the freeze-thaw behavior of water and enhancing the pore-forming effect. This results in optimal pore size distribution uniformity and channel connectivity, thereby optimizing cell adhesion and promoting cell proliferation.

[0025] According to some alternative embodiments, the amino acids also include aspartic acid and / or glutamic acid, which are negatively charged and may help keep collagen fibers in a more stretched state before freezing by electrostatic repulsion, thereby also helping to regulate pore structure.

[0026] According to some specific implementation methods, step (1) is as follows: first, dissolve the amino acids in hydrochloric acid solution. After complete dissolution, add collagen and stir at low temperature (4°C) to dissolve evenly to obtain the collagen / amino acid mixed solution. The collagen / amino acid mixed solution contains collagen with a concentration of 1~3 w / v. The amount of amino acid added is calculated based on the dry weight of collagen and is added at 5~15% of the dry weight of collagen.

[0027] According to some preferred embodiments, the nozzle diameter (outer diameter) used in the liquid nitrogen spray granulation is 0.5~2.0cm, the orifice diameter of the nozzle is 0.2mm~0.4mm, and the spray pressure is 0.2~0.4bar. It should be noted that the spray pressure of the nozzle in this invention refers to gauge pressure, which is equal to absolute pressure minus atmospheric pressure (approximately 1.01325bar). That is, if absolute pressure is used, the corresponding spray pressure is 1.21325~1.41325bar.

[0028] The present invention does not limit the specific operation of liquid nitrogen spray granulation, and those skilled in the art can make conventional choices; specifically, for example, a collagen / amino acid mixed solution is placed in a spray device, and the collagen / amino acid mixed solution is sprayed into liquid nitrogen by compressed nitrogen (or inert gas) to granulate and form tiny frozen microspheres.

[0029] In this invention, the first freeze-drying is a crucial step in pore formation. During the vacuum sublimation process of the amino acid-water system, pores are left at the locations of the crystals or eutectic formed with water. The type and proportion of amino acids directly determine the size and distribution of these "template pore-forming agents".

[0030] In this invention, the operations of the first and second freeze-drying processes can be the same or different; this invention does not impose specific limitations, and those skilled in the art can make conventional choices. In some specific embodiments of this invention, the first and second freeze-drying processes are, for example, pre-freezing at -30 to -50°C for 2 to 4 hours, then drying at -10 to -20°C for 30 to 50 hours, then drying at 2 to 10°C for 5 to 15 hours, and then drying at 20 to 30°C for 2 to 4 hours. The drying at these three temperature stages is carried out under an absolute pressure of 0 to 0.2 mbar. This invention does not impose specific limitations on the cooling rate and / or heating rate involved in the first and second freeze-drying processes; those skilled in the art can make conventional choices, for example, 0.2 to 1.5°C / min.

[0031] In this invention, the collagen microspheres obtained by the first freeze-drying process have poor mechanical strength and their structure is prone to collapse when exposed to water, so they must be cross-linked before they can be used.

[0032] According to some preferred embodiments, the crosslinking agent used is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide; the crosslinking involves placing the microspheres obtained after the first freeze-drying into a crosslinking agent solution for crosslinking, the crosslinking agent solution comprising 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and an ethanol solution; the mass-to-volume ratio (M / V) of the microspheres obtained after the first freeze-drying to the crosslinking agent solution is, for example, 1 g : (50~200) mL; preferably, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the crosslinking agent solution is [missing information]. The concentration of the crosslinking agent solution is 20-40 mmol / L; preferably, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1:(3-6); preferably, the ethanol solution is composed of anhydrous ethanol and water in a volume ratio of (6-8):(2-4); in this invention, preferably, the ethanol solution is composed of anhydrous ethanol and water in a volume ratio of (6-8):(2-4). This invention has found that if the concentration of anhydrous ethanol is too low, the collagen microspheres are easily soluble in water and collapse; if the concentration of anhydrous ethanol is too high, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride easily precipitates, and the crosslinking solution becomes cloudy instead of clear. In this invention, the crosslinking is, for example, crosslinked at 4°C for 8-16 hours, then washed three times with purified water, and then subjected to a second freeze-drying to obtain the porous collagen microspheres of this invention.

[0033] According to some preferred embodiments, the collagen is adipose-derived collagen, and the preparation of the adipose-derived collagen includes the following steps: (a) After washing the adipose tissue, it is crushed into small particles to obtain the material. Then, the material is subjected to a gradient degreasing treatment with an increasing material-to-liquid ratio using isopropanol to obtain a preliminarily degreased material. In this invention, for example, fresh adipose tissue is washed with purified water and then added to a high-speed homogenizer to be crushed into small particles with a particle size of, for example, 2-4 mm. This invention does not specifically limit the source of adipose tissue, and those skilled in the art can choose conventionally. (b) The preliminarily defatted material was decellularized using a phosphate buffer solution containing trypsin and disodium EDTA to obtain decellularized material; (c) Isopropanol was used to perform a secondary degreasing treatment on the decellularized material to obtain decellularized adipose tissue fibers; (d) First, decellularized adipose tissue fibers are treated with DNase and RNase to obtain nucleic acid-removed decellularized adipose tissue fibers. Then, the nucleic acid-removed decellularized adipose tissue fibers are soaked in an acid soaking solution before homogenization to obtain a homogenate. The homogenate is then enzymatically hydrolyzed with pepsin and neutralized. Finally, it is centrifuged to obtain a precipitate. This invention adds an acid soaking step before pepsin hydrolysis, which allows the nucleic acid-removed decellularized adipose tissue fibers to fully swell under acidic conditions, making the fiber structure loose and soft, thus significantly improving the subsequent homogenization effect. The homogenized material is a uniform and fine paste, free of... The presence of distinct particles and flocculent material facilitates uniform contact and efficient enzymatic hydrolysis between pepsin and substrate, allowing collagen to be fully released and converted into a soluble state. This process increases the collagen extraction rate, helps maintain the growth factors and collagen conformation in the ECM, and results in higher purity and more complete biological activity in the final adipose-derived collagen. It also provides an ideal microenvironment for adipocyte migration, angiogenesis, and tissue integration, thereby enhancing skin rejuvenation effects, such as promoting angiogenesis, collagen and elastin synthesis in the transplanted area, resulting in thicker, firmer, and more radiant skin. (e) The precipitate is dissolved in an acidic solution and subjected to gradient dialysis with increasing pH value. Finally, it is freeze-dried to obtain the freeze-dried product, which is the decellularized fat collagen.

[0034] The preferred embodiment of this invention is adipose-derived collagen obtained through steps (a) to (e) above. Through optimizations such as the gradient defatting strategy, pepsin hydrolysis process, and pH gradient dialysis, the key regeneration-promoting active ingredients in the adipose-derived collagen are efficiently preserved. Specifically, this invention employs a three-step progressive gradient defatting with an increasing material-to-liquid ratio, which thoroughly removes grease, avoids inflammatory reactions and barrier effects caused by residual grease, and effectively protects the collagen conformation and growth factor binding sites in the ECM. Pepsin hydrolysis minimizes the degradation loss of heat-sensitive active ingredients such as bFGF, VEGF, and TGF-β1. pH gradient dialysis effectively removes pepsin while retaining effective components such as glycosaminoglycans, while avoiding collagen aggregation and precipitation and growth factor inactivation caused by direct rapid dialysis. More importantly, the above... The active ingredients are efficiently preserved in the subsequent porous collagen microsphere preparation process of this invention. This means that the porous collagen microspheres of this invention not only provide a three-dimensional physical scaffold, but also carry bioactive regenerative signaling molecules: bFGF can promote fibroblast proliferation and collagen synthesis, which helps to restructure and improve the firmness of the skin in the transplantation area; VEGF can promote angiogenesis around the microspheres, improve blood supply, and provide necessary nutrients and oxygen for the survival of adipocytes; TGF-β1 participates in extracellular matrix synthesis and tissue repair, and promotes the production of collagen and elastin; glycosaminoglycans maintain the moisture in the microspheres and protect the activity of growth factors, while providing a good hydration environment for the tissue. The combination of these active ingredients with the porous collagen microspheres makes the product of this invention have the dual functions of physical scaffold and biological signaling, which is conducive to promoting the therapeutic effect of "skin rejuvenation".

[0035] According to some preferred embodiments, the adipose-derived collagen comprises type I collagen, type IV collagen, type V collagen, and type VI collagen. Type I collagen accounts for more than 90% of the protein mass of the adipose-derived collagen, type IV collagen accounts for 1.0-1.5% of the protein mass, type V collagen accounts for 3-3.5% of the protein mass, and type VI collagen accounts for 1.5-2.0% of the protein mass. The residual DNA content is less than 50 ng / mg. Preferably, based on the dry weight of the adipose-derived collagen, the adipose-derived collagen contains ≥100 ng / g of vascular endothelial growth factor (VEGF), ≥30 ng / g of basic fibroblast growth factor (bFGF), ≥8 ng / g of transforming growth factor-β1 (TGF-β1), and ≥1% by mass of glycosaminoglycans.

[0036] In the preferred preparation of adipose-derived collagen of this invention, type I collagen (accounting for more than 90%) provides structural support, while type IV, V, and VI collagen synergistically maintain the integrity of the basement membrane and cell anchoring sites. bFGF content ≥30 ng / g promotes fibroblast proliferation and collagen synthesis, VEGF content ≥100 ng / g promotes angiogenesis to supply oxygen to adipocytes, TGF-β1 content ≥8 ng / g participates in extracellular matrix remodeling, glycosaminoglycan content ≥1% maintains moisture and protects growth factor activity, and DNA residue is less than 50 ng / mg to ensure immune safety. The efficient retention of these active ingredients is attributed to the synergistic process of gradient defatting, low-temperature pepsin hydrolysis, and pH gradient dialysis: gradient defatting avoids damage to growth factor binding sites, low-temperature pepsin hydrolysis reduces the degradation of heat-sensitive active ingredients, and pH gradient dialysis removes pepsin while retaining glycosaminoglycans, avoiding growth factor inactivation and collagen deposition and loss due to pH mutations. This results in adipose-derived collagen with both high bioactivity and clinical usability, providing an ideal scaffold material basis for fat grafting.

[0037] According to some preferred embodiments, in step (a): the gradient degreasing involves first mixing the material with isopropanol at a material-to-liquid ratio (mass ratio) of 1:(1~5), preferably 1:(4~5), stirring at 37°C for 5~15 min, and then centrifuging once. Then, the material obtained from the first centrifugation is mixed with isopropanol at a material-to-liquid ratio (mass ratio) of 1:(1~3), preferably 1:(2~3), stirring at 37°C for 15~25 min, and then centrifuging a second time. Finally, the material obtained from the second centrifugation is mixed with isopropanol at a material-to-liquid ratio (mass ratio) of 1:(1~2), preferably 1:(1~1.5), stirring at 37°C for 25~35 min, and then centrifuging a third time. In this invention, unless otherwise specified, the stirring speed can be, for example, 100~240 rpm, the centrifugation speed can be, for example, 3000~5000 rpm, and the centrifugation time can be, for example, 3~10 min.

[0038] The present invention preferably employs the aforementioned gradient degreasing strategy. First, a three-step progressive degreasing process with a gradually changing material-to-liquid ratio is used. The first step involves high-proportion isopropanol degreasing to rapidly remove the main oil; the second step involves medium-proportion isopropanol degreasing to further remove residual oil; and the third step involves low-proportion isopropanol degreasing to remove trace amounts of oil. This approach not only facilitates thorough preliminary degreasing but also avoids the potential damage to collagen conformation and growth factor binding sites in the ECM that may be caused by traditional single high-concentration degreasing. Second, this approach is compatible with conventional methods such as repeated freeze-thaw cycles (each cycle lasting more than 24 hours, requiring 3-4 cycles), homogenization, and centrifugation for preliminary degreasing. Compared to traditional methods, the gradient degreasing technique of this invention significantly shortens the operation time (total processing time is less than 1 hour), greatly improves degreasing efficiency, and enhances the initial degreasing effect. In contrast, conventional methods of repeatedly freezing and thawing, homogenizing, and centrifuging to remove fat are inefficient and leave a significant amount of fat bound to the fiber scaffold. The gradient degreasing strategy described in this invention effectively avoids the inflammatory response and barrier effect caused by fat residue during traditional methods of repeatedly freezing and thawing, homogenizing, and centrifuging to remove fat, providing a "clean" substrate for subsequent decellularization and thus maximizing the protection of the effective active ingredients in the ECM.

[0039] According to some specific implementation methods, step (a) is as follows: fresh adipose tissue is washed with water and then added to a high-speed homogenizer to be crushed into small particles with a particle size of 3 mm to obtain material; then the material is mixed with isopropanol at a material-to-liquid ratio of 1:(1~5) and stirred (stirring speed 180~240 rpm) in a 37°C water bath for 5~15 min, and then centrifuged once at a speed of 3000~5000 rpm for 3~10 min, and the supernatant is discarded to obtain the first centrifuged material; then the first centrifuged material obtained from the first centrifugation is mixed with isopropanol at a material-to-liquid ratio of 1:(1~3). Then, start stirring (stirring speed 180~240 rpm) and stir in a 37℃ water bath for 15~25 min. Then, centrifuge for a second time at 3000~5000 rpm for 3~10 min. Discard the supernatant solution to obtain the second centrifuged material. Finally, mix the second centrifuged material with isopropanol at a material-to-liquid ratio of 1:(1~2) and start stirring (stirring speed 180~240 rpm). Stir in a 37℃ water bath for 25~35 min. Then, centrifuge for a third time at 3000~5000 rpm for 3~10 min. Discard the supernatant solution to obtain the preliminary degreased material.

[0040] According to some preferred embodiments, in step (b): the concentration of trypsin in the phosphate buffer solution is 0.01~0.1wt%, preferably 0.02wt%; the present invention does not specifically limit the phosphate buffer solution, and a phosphate buffer solution with a pH of 7.2~7.4 can be used; the mass ratio of disodium ethylenediaminetetraacetate to trypsin in the phosphate buffer solution is (1~3):(1~2), preferably 1:1.25; during the decellularization process, the mass ratio of the initially defatted material to the phosphate buffer solution is 1:(10~100); the decellularization process is carried out at a temperature of 37°C and a stirring speed of 1... The decellularization process is carried out at 00~240 rpm for 0.5~1.5 h; and / or before step (c), the process further includes squeezing the decellularized material obtained in step (b) to obtain squeezed decellularized material, and then performing a washing process, wherein the washing process includes: washing the squeezed decellularized material with water for 3~8 min and then squeezing it again, and the number of washing processes is 3~5 times; the present invention does not specifically limit the squeezing operation, and the present invention can conventionally select, for example, a juicer can be used to squeeze until there is no free-flowing water; preferably, the amount of water used in each washing is 4~8 times the mass of the squeezed decellularized material.

[0041] According to some specific implementation methods, step (b) is as follows: A phosphate buffer solution containing trypsin and disodium ethylenediaminetetraacetate (EDTA) is prepared using a phosphate buffer solution with a pH of 7.2-7.4. The concentration of trypsin in the prepared phosphate buffer solution is 0.01-0.1 wt%, and the mass ratio of disodium EDTA to trypsin is (1-3):(1-2). The preliminarily defatted material obtained in step (a) is mixed with the prepared phosphate buffer solution at a mass ratio of 1:(10-100), and the mixture is heated at 37°C. Decellularize at 100-240 rpm for 0.5-1.5 hours to obtain decellularized material. After decellularization, squeeze the decellularized material dry using a juicer to obtain squeezed decellularized material. Then, add 4-8 times the mass of the squeezed decellularized material to purified water (e.g., purified water at 3-10℃) and manually stir and wash for 3-8 minutes or at 50-80 rpm for 3-8 minutes. Squeeze dry again using a juicer. Repeat the washing and squeezing process (i.e., repeat the washing process) 3-5 times to obtain the washed decellularized material.

[0042] According to some preferred embodiments, in step (c): during the secondary defatting treatment, the mass ratio of the decellularized material to isopropanol is 1:(2~4), preferably 1:3; the secondary defatting treatment is performed in a 37°C shaking incubator at a speed of 100~240 rpm for 16~24 h; and / or before performing step (d), the method further includes squeezing the decellularized adipose tissue fibers obtained in step (c) to obtain squeezed decellularized adipose tissue fibers, and then performing a washing treatment, wherein the washing treatment includes: washing the squeezed decellularized adipose tissue fibers with water for 3~8 min and then squeezing them again, and the number of washing treatments is 3~5 times. Preferably, the amount of water used in each washing is 4~8 times the mass of the squeezed decellularized adipose tissue fibers.

[0043] The secondary degreasing in this invention effectively ensures that the residual fat content in the product is less than 1%. On the one hand, it can avoid residual oil from causing chronic inflammatory reactions or forming physical barriers at the injection site; on the other hand, after the oil is completely removed, the collagen fiber network in the ECM is more loose and open; in addition, low fat residue also significantly reduces the risk of oxidative deterioration of the product during storage and use, and prolongs the product's stability.

[0044] According to some specific implementation methods, step (c) is as follows: Isopropanol is added to the decellularized material after cleaning treatment obtained in step (b) at a mass ratio of 1:(2~4) and mixed evenly to obtain a mixture. The mixture is placed in a 37°C shaking incubator and shaken at a speed of 100~240 rpm for 16~24 hours to obtain decellularized adipose tissue fibers (DAT fibers). After the secondary defatting treatment is completed, the decellularized adipose tissue fibers are squeezed dry using a juicer to obtain squeezed decellularized adipose tissue fibers. Then, 4~8 times the mass of the squeezed decellularized adipose tissue fibers (e.g., 3~10°C purified water) is added to the squeezed decellularized adipose tissue fibers and manually stirred and washed for 3~8 minutes or stirred and washed at a speed of 50~80 rpm for 3~8 minutes. The fibers are squeezed dry again using a juicer. This washing and squeezing process (i.e., repeated washing treatment) is repeated 3~5 times to obtain cleaned decellularized adipose tissue fibers.

[0045] According to some preferred embodiments, in step (d): the treatment of decellularized adipose tissue fibers with DNase and RNase is as follows: DNase and RNase are dissolved in sodium chloride solution (sodium chloride aqueous solution) to obtain an enzyme solution, and then the decellularized adipose tissue fibers and the enzyme solution are mixed at a mass ratio of 1:(2~4), preferably 1:3, and treated with shaking at 100~240 rpm in a 37°C shaking incubator for 16~24 h; In this invention, the DNase and RNase are not specifically limited, and commercially available products can be used; In this invention, for example, DNase with an enzyme activity concentration of 1500~2000 U / mg can be used. Enzyme A is an RNase with an enzyme activity concentration of 40-60 U / mg; preferably, the sodium chloride solution contains sodium chloride at a concentration of 0.5-1.5 mol / L, preferably 1 mol / L; preferably, the amount of DNase used is 15000-25000 U (e.g., 15000, 18000, 20000, 22000 or 25000 U) of DNase per 2 kg of sodium chloride solution, and the amount of RNase used is 15000-25000 U (e.g., 15000, 18000, 20000, 22000 or 25000 U) of RNase per 2 kg of sodium chloride solution.

[0046] According to some preferred embodiments, in step (d), before soaking in the acid soaking solution, the method further includes squeezing the obtained nucleic acid-removed decellularized adipose tissue fibers to obtain squeezed nucleic acid-removed decellularized adipose tissue fibers, and then performing a washing process. The washing process includes: washing the squeezed nucleic acid-removed decellularized adipose tissue fibers with water for 3-8 minutes and then squeezing them dry again. The number of washing processes is 3-5 times. Preferably, the amount of water used in each washing is 8-15 times the mass of the squeezed nucleic acid-removed decellularized adipose tissue fibers.

[0047] According to some preferred embodiments, in step (d): the pH of the acid soaking solution is 1.8~2.2, the acid in the acid soaking solution is HCl, and the acid soaking solution also contains 3~8 wt% ethanol, that is, the mass percentage of ethanol in the acid soaking solution is 3~8%. In this invention, the acid soaking solution used for soaking preferably also contains 3~8 wt% ethanol. This invention has found that although the decellularization process removes most of the cell membrane components, lipids such as phospholipids and cholesterol may still remain in the tissue. These lipids will coat the surface of collagen fibers, forming a barrier that enzymes cannot penetrate. Ethanol is an excellent lipid solvent. This invention has found that the acid soaking solution containing 3~8 wt% ethanol can effectively dissolve and wash away these residual lipids, "cleaning" away the lipid barrier that hinders enzymatic hydrolysis, exposing the enzyme cleavage sites of collagen, compared to strong alkalis or strong oxidants, etc. The aggressive pretreatment method, using an acid soaking solution containing 3-8 wt% ethanol, is a relatively mild pretreatment system. During soaking, the dry weight of the nucleic acid-removed decellularized adipose tissue fibers to the mass ratio of the acid soaking solution is (3-6) mg:1 g. The soaking is performed at 3-10°C, preferably 4°C, for 1-2 hours. The homogenization speed is 10000-20000 rpm, preferably 15000 rpm, for 10-30 minutes. When using pepsin for enzymatic hydrolysis, the enzyme activity concentration of the pepsin is 2000-4000 U / mg, preferably 3000 U / mg, and the mass ratio of the homogenate to the pepsin is 100:(1-10). The enzymatic hydrolysis is performed at 3-10°C, preferably 4°C, for 60-96 hours. And / or the neutralization is performed to a pH of 7.0-7.2.

[0048] According to some specific implementation methods, step (d) is as follows: Weigh DNase (enzyme activity concentration 1500~2500U / mg) and RNase (enzyme activity concentration 40~60U / mg), wherein the amount of DNase is calculated based on a total enzyme activity of 15000~25000U, and the amount of RNase is calculated based on a total enzyme activity of 15000~25000U, dissolve them in a 0.5~1.5mol / L sodium chloride aqueous solution, and add 0.5~1.5mol / L sodium chloride aqueous solution to make up to 2kg to obtain an enzyme solution; take the decellularized adipose tissue fibers (DAT fibers) obtained in step (c), add them to the enzyme solution at a mass ratio of DAT fibers to enzyme solution of 1:(2~4) and mix well, then place them in a 37℃ shaking incubator, and incubate at 100~24... The fibers were treated with oscillation at 0 rpm for 16-24 hours to obtain nucleic acid-removed decellularized adipose tissue fibers. After treatment, the fibers were squeezed dry using a juicer to obtain squeezed nucleic acid-removed decellularized adipose tissue fibers. Then, 8-15 times the weight of the squeezed nucleic acid-removed decellularized adipose tissue fibers (e.g., purified water at 20-30℃) were added to the squeezed fibers and manually stirred and washed for 3-8 minutes, or stirred and washed at 50-80 rpm for 3-8 minutes. The fibers were squeezed dry again using a juicer. This washing and squeezing process (i.e., repeated washing process) was repeated 3-5 times to obtain cleaned nucleic acid-removed decellularized adipose tissue fibers. These were then placed in sterile bags and stored at 2-8℃. The cleaned nucleic acid-removed decellularized adipose tissue fiber samples were taken and measured using an infrared rapid moisture analyzer at 105℃ for 30 minutes. The moisture content was recorded for use in the calculation of sample dry weight in subsequent steps. An acid soaking solution was prepared using purified water, anhydrous ethanol, and concentrated hydrochloric acid (e.g., 36-38 wt% concentrated hydrochloric acid) to obtain an acid soaking solution with a pH of 1.8-2.2 and containing 3-8 wt% ethanol. The cleaned and treated nucleic acid-removed decellularized adipose tissue fibers obtained above were weighed, and the dry weight of the cleaned and treated nucleic acid-removed decellularized adipose tissue fibers was calculated based on the moisture content test results. The cleaned and treated nucleic acid-removed decellularized adipose tissue fibers were mixed with the acid soaking solution at a mass ratio of (3-6) mg:1 g. The sample was homogenized and soaked at 3-10℃ for 1-2 hours. Then, the sample soaked in the acid soaking solution was homogenized using a high-shear homogenizer at a speed of 10,000-20,000 rpm for 10-30 minutes to obtain a homogenate. The amount of pepsin was calculated according to the mass ratio of homogenate to pepsin of 100:(1-10) (pepsin activity concentration of 2000-4000 U / mg). The pepsin was added to the homogenate and mixed evenly. Then, the sample was placed in a shaking incubator at 3-10℃ for 60-96 hours to obtain a pepsin hydrolysate.After enzymatic hydrolysis, the pH of the pepsin hydrolysate was adjusted to 7.0-7.2 using an aqueous sodium hydroxide solution (e.g., a sodium hydroxide solution with a concentration of 0.05-0.2 mol / L) and allowed to stand for 20-40 minutes. Finally, the sample was centrifuged at 8000 rpm for 20 minutes using a refrigerated centrifuge, the supernatant was discarded, and the precipitate was collected. The present invention does not impose specific limitations on the operation of centrifugation using a refrigerated centrifuge, and those skilled in the art can choose conventionally.

[0049] According to some preferred embodiments, in step (e): the acid solution is a hydrochloric acid solution with a concentration of 0.0005~0.02mol / L; the precipitate is dissolved in the acid solution to obtain dialysate, and then the dialysate is loaded into a dialysis bag with a molecular weight cutoff of 35~100kDa for gradient dialysis. The gradient dialysis is performed by first using a hydrochloric acid solution with a pH of 2.8~3.2 as the dialysate for one day, then using a hydrochloric acid solution with a pH of 4.8~5.2 as the dialysate for one day, and finally using water as the dialysate for one day. Preferably, the mass ratio of dialysate to dialysate is 1:(10~20). Preferably, the temperature of the dialysate and dialysate is maintained at 4~10°C during dialysis.

[0050] The present invention preferably uses dialysis bags with a molecular weight cutoff of 35kDa to 100kDa, more preferably 50kDa. The present invention has found that pepsin has a molecular weight of about 35kDa, which can be effectively removed by freely passing through the dialysis bag, while active ingredients such as glycosaminoglycans are retained due to their larger molecular weight, thereby achieving the protection of active ingredients while purifying collagen. Existing technologies pay little attention to the removal effect of additives such as pepsin. This invention solves this problem by optimizing the molecular weight cutoff of the dialysis bag. In addition, this invention preferably uses the above-mentioned pH gradient dialysis, which has a synergistic effect: On the one hand, it can promote the orderly refolding of collagen. Collagen molecules fully dissolve and unfold into single-stranded peptide chains under low pH conditions (pH about 2). If they are directly transferred to a neutral pH environment, the peptide chains will instantly begin to fold and approach each other, which can easily lead to accidental collisions and aggregation, resulting in collagen "explosion" and forming a heterogeneous mixture of partial dissolution, partial gelation, and partial precipitation. This invention provides each collagen peptide chain with sufficient time and a gentle environment by slowly and gradually increasing the pH, allowing it to fold and assemble in an orderly and gradual manner, greatly reducing the chance of accidental collisions and aggregation between molecules, and finally obtaining a clear, homogeneous, and functionally complete collagen solution. On the other hand, it is beneficial for microbial control during the dialysis process. Collagen is a natural culture medium and is extremely prone to the growth of microorganisms in a neutral environment. This invention maintains low pH conditions (pH 2.8~5.2) during the early and middle stages of gradient dialysis, which has a highly efficient killing effect on most microorganisms, effectively controlling the risk of microbial contamination during dialysis and ensuring product safety. In addition, the gradient dialysis used in this invention can also avoid growth factor inactivation caused by pH changes and loss of growth factors caused by collagen precipitation. By optimizing the molecular weight cutoff of the dialysis bag and combining it with optimized pH gradient dialysis, this invention achieves effective removal of pepsin, retention of active ingredients such as glycosaminoglycans and growth factors, orderly collagen, and microbial control during dialysis, which is beneficial for preparing high-purity, high-bioactivity, uniform and stable adipose-derived collagen.

[0051] According to some specific implementation methods, step (e) is as follows: the precipitate obtained in step (d) is dissolved in a hydrochloric acid solution with a concentration of 0.0005~0.02mol / L to obtain a dialysate, and the mass ratio of precipitate to acid solution is 1:(10~50); then the dialysate is placed into a dialysis bag with a molecular weight cutoff of 35kDa~100kDa, the two ends are clamped, and the bag is placed in a dialysis tank for gradient dialysis. The gradient dialysis is performed by first using a hydrochloric acid solution with a pH of 2.8~3.2 as the dialysate for one day (24h), then using a hydrochloric acid solution with a pH of 4.8~5.2 as the dialysate for one day (24h), and finally using purified water as the dialysate for one day (24h); during dialysis, the mass ratio of dialysate to dialysate is 1:(10~20), the dialysate is changed twice a day, and the temperature of the dialysate and dialysate is maintained at 4~ 10℃; The solution obtained after gradient dialysis is placed into a stainless steel freeze-drying tray, the freeze-drying tray is covered with a medical packaging bag (e.g., a Tyvek bag), sealed, and then placed in a freeze dryer for freeze-drying to obtain the freeze-dried product, which is the adipose-derived collagen. The present invention does not specifically limit the parameters of the freeze-drying involved in this step, and those skilled in the art can choose conventionally. In some specific embodiments of the present invention, the freeze-drying is, for example, first pre-freezing at -40~-50℃ for 2~4h, then drying at -10~-30℃ for 40~60h, and finally drying at 5~20℃ for 5~15h. The entire freeze-drying is carried out under an absolute pressure of 10~1000μbar. The present invention does not specifically limit the cooling rate and / or heating rate involved in the freeze-drying process, and those skilled in the art can choose conventionally, for example, 0.2~1.5℃ / min.

[0052] The present invention provides, in a second aspect, porous collagen microspheres prepared by the preparation method described in the first aspect of the present invention.

[0053] In a third aspect, the present invention provides the application of porous collagen microspheres prepared by the preparation method described in the first aspect of the present invention as core materials for tissue engineering scaffolds, cell culture carriers, drug delivery carriers, or core-shell structured microspheres; preferably, the core-shell structured microspheres are dual growth factor sustained-release microspheres with a core-shell structure; preferably, the dual growth factors are platelet-derived growth factor PDGF-BB and vascular endothelial growth factor VEGF.

[0054] This invention reveals that the porous collagen microspheres described herein are particularly suitable for use as core materials in tissue engineering scaffolds, cell culture carriers, drug delivery carriers, or core-shell microspheres, and more preferably as core materials in core-shell microspheres. Specifically, for example, core-shell microspheres prepared by loading PDGF-BB onto the porous collagen microspheres of this invention as the core material and then coating them with a VEGF-collagen shell using microfluidic technology exhibit excellent time-sequential release properties, forming a time-sequential release system of "rapid release of VEGF from the shell / slow release of PDGF-BB from the core," thus mimicking angiogenesis. The physiological process (first VEGF induces budding, then PDGF-BB promotes maturation) provides an ideal artificial ECM microenvironment for fat grafting. The porous collagen microspheres of this invention are perfectly suited for preparing core-shell structured dual growth factor sustained-release microspheres. They are the core material for the latter to achieve the biochemical / mechanical properties and time-sequential release function of mimicking natural ECM. They can sustainably release vascular endothelial growth factor and platelet-derived growth factor, promote the successful establishment of blood supply in fat cells, and provide an ideal artificial ECM scaffold for fat grafting, solving the problem of fat cell ischemia and necrosis, and ultimately contributing to the therapeutic effect of "skin rejuvenation".

[0055] In this invention, when the porous collagen microspheres are used as the core material of dual growth factor sustained-release microspheres with a core-shell structure, platelet-derived growth factor (PDGF-BB) is added to the collagen / amino acid mixed solution during preparation. The amount of PDGF-BB added is such that the concentration of PDGF-BB in the collagen / amino acid mixed solution is 10~50 μg / mL. Then, the collagen / amino acid mixed solution with added platelet-derived growth factor is subjected to liquid nitrogen spray granulation, first freeze-drying, cross-linking, and second freeze-drying to obtain porous collagen microspheres encapsulated with PDGF-BB (platelet-derived growth factor). PDGF-BB (platelet-derived growth factor) is encapsulated in the cross-linked porous collagen microspheres.

[0056] In this invention, the preparation of the core-shell structured dual growth factor sustained-release microspheres includes, for example, the following steps: S1. Dilute the collagen solution obtained after gradient dialysis in step (e) with pre-cooled (e.g., pre-cooled to 4°C) MES buffer to a collagen concentration of 0.8–1 mg / mL. The entire process should be performed in an ice bath or at 4°C to prevent premature gelation of the collagen. Then adjust the pH of the system to 5.5 to obtain the collagen MES buffer. Before use, prepare a 300–500 mM EDC hydrochloride stock solution and a 50–150 mM NHS stock solution using the pre-cooled MES buffer. Add the NHS stock solution and EDC hydrochloride stock solution sequentially to the collagen MES buffer, bringing the final NHS concentration to 2.5–10 mM and the final EDC hydrochloride concentration to 10–40 mM, and mix thoroughly to obtain the cross-linking reaction. The system involves dissolving vascular endothelial growth factor (VEGF) in pre-cooled MES buffer (e.g., a VEGF to MES buffer mass ratio of 1:(1~2)), adding it to the cross-linking reaction system within 10 min to achieve a final VEGF concentration of 1~10 μg / mL, and reacting at 4°C for 2-6 h, for example, by shaking at low speed (e.g., 30~80 rpm) in a shaker at 4°C for 2-6 h. In this invention, by shortening the cross-linking reaction time and the concentration of the cross-linking agents (NHS and EDC hydrochloride), the cross-linking reaction system is optimized. The system retains its fluidity after the cross-linking reaction. Subsequently, a 0.1M Na₂HPO₄ aqueous solution or PBS buffer is added, with a volume of 1 / 10 to 1 / 5 of the total volume of the cross-linking reaction system. The mixture is incubated at 4°C for 2-4 hours to quench unreacted EDC hydrochloride and / or NHS and hydrolyze unstable intermediates, yielding a reaction mixture. The reaction mixture is then ultrafiltered through an ultrafiltration membrane to remove byproducts (e.g., urea derivatives) and ungrafted VEGF. The mixture is then concentrated using the concentration system of the ultrafiltration equipment to a VEGF-grafted collagen mass-volume concentration of 0.8-1 w / v%, yielding a VEGF-grafted collagen solution. In this invention, the unit "mM" stands for "mmol / L". In this invention, pre-cooling is, for example, pre-cooling to 4°C. This invention does not specifically limit the molecular weight cutoff of the ultrafiltration membrane; those skilled in the art can choose conventionally, for example, a molecular weight cutoff of 10-70 kDa, preferably 40-70 kDa. This invention does not specifically limit the MES buffer; any commercially available product can be used. S2. The porous collagen microspheres encapsulated with PDGF-BB obtained in this invention are suspended in PBS buffer at a concentration of 1-10 mg / mL as the inner phase (also referred to as the core suspension); the collagen solution grafted with VEGF is used as the intermediate phase (shell precursor solution); 0.5-2 wt% Span is added. Mineral oil of grade 80 (Span 80) was used as the external phase (oil phase). A coaxial microfluidic chip was used with three inlets connected to the internal phase, intermediate phase, and external phase respectively. The flow rates were controlled at 1-10 μL / min for the internal phase, 5-20 μL / min for the intermediate phase, and 100-500 μL / min for the external phase, generating stable core-shell W / O / W droplets in the flow focusing region. The core-shell W / O / W droplets were collected in mineral oil containing 0.1-0.5 wt% glutaraldehyde for cross-linking and curing (e.g., room temperature cross-linking and curing for 2-4 hours), or the core-shell W / O / W droplets were collected and then subjected to 365 nm ultraviolet light (UV irradiation intensity of 5-15 mW / cm²). 2 Photocrosslinking and curing are performed by irradiation for 30-60 seconds to obtain core-shell structured microspheres. The core-shell structured microspheres are then centrifuged (1000-2000 rpm, 5 minutes) to remove the oil phase, washed 2-3 times with n-hexane or hexane, and then washed 3-5 times with PBS buffer. The washed microspheres are then freeze-dried to obtain dual growth factor sustained-release microspheres with a core-shell structure (also referred to as artificial extracellular matrix). In this invention, the room temperature is, for example, 15-35°C. This invention does not impose specific limitations on the mineral oil used; those skilled in the art can choose conventionally.

[0057] As is well known, the main challenges of autologous fat transplantation are: fat cells are large, fragile, and sensitive to hypoxia. They require rapid establishment of blood supply within 200 micrometers after transplantation to survive; otherwise, they will necrose due to ischemia. Simply injecting fat masses often results in the lack of vascular support in the central area, making survival difficult. Therefore, this invention preferably combines core-shell structured dual growth factor sustained-release microspheres with fat cells (e.g., autologous fat cells). Through the physical support of the core-shell structured dual growth factor sustained-release microsphere scaffold and the time-sequential release of growth factors, a three-dimensional microenvironment mimicking the natural ECM is provided for the fat cells.

[0058] Specific procedures include: adding the obtained adipose tissue to physiological saline and centrifuging at 1200-1500 rpm for 1-3 minutes, discarding the upper layer of oil and the lower layer of blood, and collecting the middle layer of purified fat. Adding physiological saline or lactated Ringer's solution to the collected purified fat for washing, centrifuging under the same conditions, and discarding the supernatant, repeating the washing and centrifugation 1-2 times to obtain the processed adipocytes. This process further removes impurities, and stirring and washing for 1-2 minutes is sufficient to avoid over-processing (e.g., prolonged exposure to air) and damage to cell viability. The processed adipocytes are then mixed evenly with the core-shell structured dual growth factor sustained-release microspheres to obtain the fat-microsphere complex. In this invention, the amount of physiological saline or lactated Ringer's solution used for washing is, for example, 3-10 times the volume of the purified fat, and the amount of the processed adipocytes is, for example, 1-10% of the mass of the core-shell structured dual growth factor sustained-release microspheres.

[0059] When the core-shell dual-growth factor sustained-release microspheres of this invention are used in combination with autologous fat cells, after injection into the body, the VEGF grafted to the outer shell of the core-shell dual-growth factor sustained-release microspheres is rapidly released, inducing vascular endothelial cell budding and initiating angiogenesis; the PDGF-BB encapsulated in the inner core is slowly released, recruiting pericytes and smooth muscle cells to encapsulate the newly formed blood vessels, promoting stable vascular maturation. The synergistic effect of the sequential release of the two growth factors rapidly establishes functional blood supply around the fat cells, improving fat survival rate and ultimately achieving skin rejuvenation, such as increased skin thickness, firmness, and radiance. In contrast, if growth factors are not loaded (only blank core-shell microspheres), angiogenesis cannot be effectively induced, and fat cells are difficult to survive due to ischemia; if VEGF and PDGF-BB are simply physically mixed and loaded, the release behavior of the two growth factors tends to converge, failing to form a sequential release system of "rapid release of outer shell VEGF / slow release of inner core PDGF-BB," making it difficult to achieve an effective connection between vascular budding and maturation, and significantly reducing angiogenesis efficiency.

[0060] The present invention will be further described below by way of examples, but the scope of protection of the present invention is not limited to these embodiments. The present invention may have many other embodiments, and those skilled in the art can make various corresponding changes and modifications based on the present invention without departing from its spirit and essence. However, all such corresponding changes and modifications should fall within the scope of protection of the appended claims. Unless otherwise specified, the experimental methods used in the following embodiments and comparative examples are conventional methods. Unless otherwise specified, the materials used in the following embodiments and comparative examples are commercially available.

[0061] Example 1 ① Fresh pork fat tissue was washed with purified water and crushed into small particles with a diameter of 3 mm using a high-speed homogenizer to obtain the material. Then, the material was mixed with isopropanol at a material-to-liquid ratio (mass ratio) of 1:4.5 and stirred (stirring speed 200 rpm) in a 37℃ water bath for 10 min. After stirring, it was centrifuged once at 4000 rpm for 5 min. The supernatant was discarded to obtain the first centrifuged material. Then, the first centrifuged material was mixed with isopropanol at a material-to-liquid ratio (mass ratio) of 1:2. 5. Mix and start stirring (stirring speed 200 rpm). After stirring in a 37℃ water bath for 20 min, centrifuge again at 4000 rpm for 5 min. Discard the supernatant solution to obtain the second centrifuged material. Finally, mix the second centrifuged material with isopropanol at a material-to-liquid ratio of 1:1.5 and start stirring (stirring speed 200 rpm). After stirring in a 37℃ water bath for 30 min, centrifuge a third time at 4000 rpm for 5 min. Discard the supernatant solution to obtain the preliminary degreased material.

[0062] ② A phosphate buffer solution containing trypsin and disodium EDTA was prepared using a phosphate buffer solution with a pH of 7.2. The concentration of trypsin in the prepared phosphate buffer solution was 0.02 wt%, and the mass ratio of disodium EDTA to trypsin was 4:5. The preliminarily defatted material obtained in step ① was mixed with the prepared phosphate buffer solution at a mass ratio of 1:50, and decellularized at 37°C and 120 rpm for 1 hour to obtain decellularized material. After decellularization, the decellularized material was squeezed dry using a juicer to obtain squeezed decellularized material. Then, purified water at 4°C, five times the mass of the squeezed decellularized material, was added to the squeezed decellularized material and stirred and washed at 60 rpm for 5 minutes. The material was squeezed dry again using a juicer. This washing and squeezing process (i.e., repeated washing process) was repeated 4 times to obtain the washed decellularized material.

[0063] ③ Add isopropanol to the decellularized material obtained in step ② at a mass ratio of 1:3 and mix evenly to obtain a mixture. Place the mixture in a 37℃ shaking incubator and shake at 180 rpm for 16 hours (secondary degreasing treatment) to obtain decellularized adipose tissue fibers (DAT fibers). After the secondary degreasing treatment, use a juicer to squeeze the decellularized adipose tissue fibers to obtain squeezed decellularized adipose tissue fibers. Then add 5 times the mass of the squeezed decellularized adipose tissue fibers in purified water at 4℃ to the squeezed decellularized adipose tissue fibers and stir and wash at 60 rpm for 5 minutes. Squeeze dry again using a juicer. Repeat the washing and squeezing process (i.e., repeat the washing process) 4 times to obtain the cleaned decellularized adipose tissue fibers.

[0064] ④ Weigh out DNase (enzyme activity concentration 2000 U / mg) and RNase (enzyme activity concentration 50 U / mg), where the amount of DNase is based on a total enzyme activity of 20000 U and the amount of RNase is based on a total enzyme activity of 20000 U. Dissolve them in a 1 mol / L sodium chloride aqueous solution, with a total sodium chloride aqueous solution volume of 2 kg, to obtain an enzyme solution. Take the decellularized adipose tissue fibers (DAT fibers) obtained in step ③ after washing and treatment, add them to the enzyme solution at a mass ratio of DAT fibers to enzyme solution of 1:3 and mix well. Then place the mixture in a 37℃ shaking incubator and shake at 180 rpm for 24 hours. h, nucleic acid-removed decellularized adipose tissue fibers were obtained. After processing, the nucleic acid-removed decellularized adipose tissue fibers were squeezed dry using a juicer to obtain squeezed nucleic acid-removed decellularized adipose tissue fibers. Then, 10 times the weight of the squeezed nucleic acid-removed decellularized adipose tissue fibers (purified water at 25℃) were added to the squeezed nucleic acid-removed decellularized adipose tissue fibers, and the mixture was stirred and washed at 60 rpm for 5 minutes. The juicer was squeezed dry again, and this washing and squeezing process was repeated 5 times to obtain cleaned nucleic acid-removed decellularized adipose tissue fibers. These were then placed in sterile bags and stored at 4℃. The cleaned nucleic acid-removed decellularized adipose tissue fiber samples were taken and the moisture content was measured using an infrared rapid moisture analyzer at 105℃ for 30 minutes. The moisture content was recorded for use in the calculation of the dry weight of the samples in subsequent steps. An acid soaking solution was prepared using purified water, anhydrous ethanol, and 36 wt% concentrated hydrochloric acid to obtain an acid soaking solution with a pH of 2.0 and containing 5 wt% ethanol. The cleaned and treated acellular adipose tissue fibers were weighed, and their dry weight was calculated based on moisture content. The dry weight of the acellular adipose tissue fibers was mixed with the acid soaking solution at a mass ratio of 4 mg:1 g, and the mixture was soaked at 4°C for 1.5 h. The sample soaked in the acid soaking solution was homogenized using a high-shear homogenizer at 15000 rpm for 20 min to obtain a homogenate. The amount of pepsin was calculated based on a mass ratio of 100:4 (pepsin activity concentration 3000 U / mg) and added to the homogenate. The mixture was then placed in a 4°C shaking incubator for 72 h to obtain a pepsin-hydrolyzed sample. After enzymatic hydrolysis, the pH of the pepsin hydrolysate was adjusted to 7.2 using a 0.1 mol / L sodium hydroxide aqueous solution and allowed to stand for 30 min. Finally, the sample was centrifuged at 8000 rpm for 20 min using a refrigerated centrifuge, the supernatant was discarded, and the precipitate was collected to obtain the precipitate.

[0065] ⑤ Dissolve the precipitate obtained in step ④ in a 0.01 mol / L hydrochloric acid solution (acid solution) to obtain a clear dialysate. The mass ratio of precipitate to hydrochloric acid solution is 1:25. Then, load the dialysate into a dialysis bag with a molecular weight cutoff of 50 kDa, clamp both ends, and place it in a dialysis tank for gradient dialysis. The gradient dialysis involves first using a hydrochloric acid solution with a pH of 3.0 as the dialysate for one day, then using a hydrochloric acid solution with a pH of 5.0 as the dialysate for one day, and finally using purified water as the dialysate for one day. During dialysis, the mass ratio of dialysate to dialysate is 1:10, and the dialysate is changed twice a day. During dialysis, the temperature of the dialysate and dialysate water is maintained at 4°C. The solution obtained after gradient dialysis is placed in a stainless steel freeze-drying tray, which is then covered with a medical packaging bag, sealed, and placed in a freeze dryer for freeze-drying. The freeze-dried product is the adipose-derived collagen. The freeze-drying process involves first cooling from room temperature to -45°C (uniform cooling rate, cooling time 1 hour) and pre-freezing at -45°C for 3 hours, then heating to -10°C (uniform heating rate, heating time 1 hour) and freeze-drying at -10°C for 48 hours, and finally heating to 10°C and drying at 10°C for 10 hours. The entire freeze-drying process is carried out under an absolute pressure of 200 μbar.

[0066] ⑥ First, dissolve the amino acids in a 0.01 mol / L hydrochloric acid solution. After complete dissolution, add the adipose-derived collagen obtained in step ⑤ and stir at a low temperature of 4°C until homogeneous to obtain a collagen / amino acid mixed solution. The concentration of adipose-derived collagen in the collagen / amino acid mixed solution is 2 w / v, and the mass of amino acids is 10 wt% of the dry weight of adipose-derived collagen. The amino acids are composed of glycine, proline, and hydroxyproline in a mass ratio of 2:1:1.

[0067] ⑦ Place the collagen / amino acid mixed solution obtained in step ⑥ into a spray device, and spray the collagen / amino acid mixed solution into liquid nitrogen by compressed nitrogen gas to perform liquid nitrogen spray granulation to obtain frozen microspheres; the nozzle diameter used for liquid nitrogen spray granulation is 1 cm, the orifice diameter of the nozzle is 0.2 mm, and the spray pressure is 0.3 bar.

[0068] ⑧ The frozen microspheres obtained in step ⑦ are subjected to a first freeze-drying, cross-linking, and a second freeze-drying in sequence to prepare porous collagen microspheres; the first and second freeze-drying processes are as follows: first, the temperature is lowered from room temperature to -30℃ (uniform cooling rate, cooling time 1h) and pre-frozen at -30℃ for 3h; then, the temperature is raised to -10℃ (uniform heating rate, heating time 1h) and dried at -10℃ for 40h (absolute pressure during drying is 0.2mbar); then, the temperature is raised to 4℃ (uniform heating rate, heating time 1h) and dried at 4℃ for 10h (absolute pressure during drying is 0.1mbar); then, the temperature is raised to 25℃ (uniform heating rate, heating time 1h) and dried at 25℃ for 3h (absolute pressure during drying is 0.1mbar). (For a pressure of 0.001 mbar); the crosslinking process involves placing the microspheres obtained after the first freeze-drying into a crosslinking agent solution for crosslinking. The crosslinking agent solution contains 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and an ethanol solution; the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the crosslinking agent solution is 30 mmol / L; the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide in the crosslinking agent solution is 1:4; the ethanol solution consists of anhydrous ethanol and water in a volume ratio of 7:3; the crosslinking process is performed at 4°C for 12 h.

[0069] The HE staining results of decellularized collagen in adipose tissue in this embodiment are shown in the image below. Figure 1 As shown, from Figure 1 The results showed no intact cell nuclei; the Oil Red O staining results of decellularized collagen in this example are shown in the image below. Figure 2 As shown, Figure 2 The results showed that Oil Red O staining was negative, indicating complete defatting. The surface morphology of the porous collagen microspheres prepared in this embodiment was observed using scanning electron microscopy (SEM), as shown in the image. Figure 3 As shown in the figure; the porous collagen microspheres prepared in this embodiment were observed using scanning electron microscopy (SEM) to reveal the cross-sectional morphology of the internal porous structure of the microspheres. Figure 4 As shown; in this embodiment, the median particle size D50 of the porous collagen microspheres was 52 μm, and the D90 was less than 100 μm, as observed by scanning electron microscopy and measured by ImageJ image analysis software. The average pore size of the porous structure inside the microspheres was 6 ± 0.5 μm. The particle size distribution of the porous collagen microspheres prepared in this embodiment is beneficial to effectively avoid being phagocytosed by macrophages, while also reducing the risk of granulomas or nodules.

[0070] The present invention also analyzed the collagen type and content of the adipose-derived collagen in this embodiment. Liquid chromatography-tandem mass spectrometry was used to identify the characteristic polypeptides of different collagen types using high-resolution mass spectrometry, and the relative percentage content of each collagen (such as type I, type IV, type V, type VI, etc.) was calculated by intensity normalization method. The results are shown in Table 1.

[0071] This invention also employs the 1,9-dimethylmethylene blue colorimetric method with a glycosaminoglycan detection kit to determine that the total glycosaminoglycan mass percentage of the adipose-derived collagen obtained in this embodiment is 1.2%. This invention also uses enzyme-linked immunosorbent assay (ELISA) to detect vascular endothelial growth factor, basic fibroblast growth factor, and transforming growth factor-β1, using specific ELISA kits for each. The absorbance of the standard and sample was measured at 450 nm using a double-antibody sandwich method, and the content of each growth factor in the sample was quantitatively calculated (unit: ng / g dry weight matrix). The vascular endothelial growth factor (VEGF) content of the adipose-derived collagen prepared in this embodiment was found to be 152 ng / g (dry weight matrix), the basic fibroblast growth factor (bFGF) content was 45.2 ng / g (dry weight matrix), and the transforming growth factor-β1 (TGF-β1) content was 8.7 ng / g (dry weight matrix). The residual DNA content in the sample was determined by real-time quantitative PCR, and the residual DNA content of the adipose-derived collagen prepared in this embodiment was found to be 24.26 ng / mg, which is lower than the standard limit of 50 ng / mg. In addition, the fat content in the adipose-derived collagen prepared in this embodiment was only 0.5% after Oil Red O staining and quantitative analysis.

[0072] Table 1 The results above show that the adipose-derived collagen raw material in this embodiment effectively retains type I, IV, and VI collagen, as well as various regenerative active ingredients such as bFGF, VEGF, TGF-β1, and glycosaminoglycans. Simultaneously, DNA residue is below 50 ng / mg, and defatting is complete. This high retention of active ingredients allows the resulting porous collagen microspheres to inherit the bioactivity of the raw material, exhibiting low immunogenicity, a suitable physical scaffold, and abundant regenerative signals. This provides an ideal scaffold for fat grafting, promotes angiogenesis and tissue integration, and is more conducive to achieving skin rejuvenation.

[0073] Example 2 Example 2 is basically the same as Example 1, except that: The amino acid used in step ⑥ is composed of glycine, hydroxyproline and glutamine in a mass ratio of 2:1:1.

[0074] Example 3 Example 3 is basically the same as Example 1, except that: The amino acid used in step ⑥ is glycine.

[0075] Example 4 Example 4 is basically the same as Example 1, except that: In step ⑥, the mass of amino acids used is 5% of the dry weight of decellularized adipose collagen.

[0076] Example 5 Example 5 is basically the same as Example 1, except that: In step ⑥, the mass of amino acids used is 15% of the dry weight of decellularized adipose collagen.

[0077] Example 6 Example 6 is basically the same as Example 1, except that: In step ⑥, the amount of amino acids used is 3% of the dry weight of decellularized adipose collagen.

[0078] Example 7 Example 7 is basically the same as Example 1, except that: In step ⑥, the amount of amino acids used is 20% of the dry weight of the decellularized adipose collagen.

[0079] Example 8 Example 8 is basically the same as Example 1, except that: ① Fresh pork fat tissue was washed with purified water and crushed into small particles with a diameter of 3 mm using a high-speed homogenizer to obtain the material. Then, the material was mixed with isopropanol at a material-to-liquid ratio (mass ratio) of 1:4.5 and stirred (stirring speed 200 rpm) in a 37℃ water bath for 10 min. After stirring, it was centrifuged once at 4000 rpm for 5 min. The supernatant was discarded to obtain the first centrifuged material. Then, the first centrifuged material was mixed with isopropanol at a material-to-liquid ratio (mass ratio) of 1:4. 5. Mix and start stirring (stirring speed 200 rpm). After stirring in a 37℃ water bath for 20 min, centrifuge again at 4000 rpm for 5 min. Discard the supernatant to obtain the second centrifuged material. Finally, mix the second centrifuged material with isopropanol at a material-to-liquid ratio of 1:4.5 and start stirring (stirring speed 200 rpm). After stirring in a 37℃ water bath for 30 min, centrifuge a third time at 4000 rpm for 5 min. Discard the supernatant to obtain the preliminary degreased material.

[0080] The basic fibroblast growth factor (bFGF) content of the adipose-derived collagen in this embodiment was measured to be 40.8 ng / g (dry weight matrix).

[0081] Example 9 Example 9 is basically the same as Example 1, except that: ① Fresh pork fat tissue was washed with purified water and crushed into small particles with a diameter of 3 mm using a high-speed homogenizer to obtain the material. Then, the material was mixed with isopropanol at a material-to-liquid ratio (mass ratio) of 1:1.5 and stirred (stirring speed 200 rpm) in a 37℃ water bath for 10 min. After stirring, it was centrifuged once at 4000 rpm for 5 min. The supernatant was discarded to obtain the first centrifuged material. Then, the first centrifuged material was mixed with isopropanol at a material-to-liquid ratio (mass ratio) of 1:1. 5. Mix and start stirring (stirring speed 200 rpm). After stirring in a 37℃ water bath for 20 min, centrifuge again at 4000 rpm for 5 min. Discard the supernatant solution to obtain the second centrifuged material. Finally, mix the second centrifuged material with isopropanol at a material-to-liquid ratio of 1:1.5 and start stirring (stirring speed 200 rpm). After stirring in a 37℃ water bath for 30 min, centrifuge a third time at 4000 rpm for 5 min. Discard the supernatant solution to obtain the preliminary degreased material.

[0082] The basic fibroblast growth factor (bFGF) content of the adipose-derived collagen in this embodiment was measured to be 35.1 ng / g (dry weight matrix).

[0083] Example 10 Example 10 is basically the same as Example 1, except that: ④ Weigh out DNase (enzyme activity concentration 2000U / mg) and RNase (enzyme activity concentration 50U / mg), where the amount of DNase is based on a total enzyme activity of 20000U and the amount of RNase is based on a total enzyme activity of 20000U. Dissolve them in 1mol / L sodium chloride aqueous solution (2kg of sodium chloride aqueous solution) to obtain an enzyme solution. Take the decellularized adipose tissue fibers (DAT fibers) obtained in step ③ after washing and treatment, add them to the enzyme solution at a mass ratio of DAT fibers to enzyme solution of 1:3 and mix well. Then place the mixture in a 37℃ shaking incubator and shake at 180rpm for 24h to obtain nucleic acid-removed decellularized adipose tissue fibers. After treatment, use a juicer to remove the nucleic acid and decellularize the fibers. The adipose tissue fibers were squeezed dry to obtain decellularized adipose tissue fibers with nucleic acid removed. Then, 10 times the weight of the decellularized adipose tissue fibers with nucleic acid removed were added to the fibers and the mixture was stirred and washed at 60 rpm for 5 minutes. The fibers were then squeezed dry again using a juicer. This washing and squeezing process (i.e., repeated washing process) was repeated 5 times to obtain cleaned decellularized adipose tissue fibers with nucleic acid removed. These fibers were then placed in sterile bags and stored at 4°C. The cleaned decellularized adipose tissue fiber samples were taken and measured using an infrared rapid moisture analyzer at 105°C for 30 minutes. The moisture content was recorded for use in the calculation of the dry weight of the samples in subsequent steps. An acid soaking solution with a pH of 2.0 was prepared using purified water and 36wt% concentrated hydrochloric acid. The cleaned and decellularized adipose tissue fibers were weighed, and their dry weight was calculated based on moisture content. The dry weight of the fibers was mixed with the acid soaking solution at a ratio of 4 mg:1 g, and the mixture was soaked at 4°C for 1.5 h. The acid-soaked sample was then homogenized using a high-shear homogenizer at 15000 rpm for 20 min to obtain a homogenate. The amount of pepsin was calculated based on a homogenate-to-pepsin ratio of 100:4 (pepsin activity concentration 3000 U / mg). The pepsin was added to the homogenate and mixed thoroughly. The mixture was then placed in a 4°C shaking incubator for 72 h to obtain a pepsin-hydrolyzed sample. After enzymatic hydrolysis, the pH of the pepsin hydrolysate was adjusted to 7.2 using a 0.1 mol / L sodium hydroxide aqueous solution and allowed to stand for 30 min. Finally, the sample was centrifuged at 8000 rpm for 20 min using a refrigerated centrifuge, the supernatant was discarded, and the precipitate was collected to obtain the precipitate.

[0084] Example 11 Example 11 is basically the same as Example 1, except that: ④ Weigh out DNase (enzyme activity concentration 2000 U / mg) and RNase (enzyme activity concentration 50 U / mg), where the amount of DNase is based on a total enzyme activity of 20000 U and the amount of RNase is based on a total enzyme activity of 20000 U. Dissolve them in a 1 mol / L sodium chloride aqueous solution (2 kg of sodium chloride aqueous solution) to obtain an enzyme solution. Take the decellularized adipose tissue fibers (DAT fibers) obtained in step ③ after washing and treatment, add them to the enzyme solution at a mass ratio of DAT fibers to enzyme solution of 1:3 and mix well. Then place the mixture in a 37℃ shaking incubator and shake at 180 rpm for 24 hours. Nucleic acid-removed decellularized adipose tissue fibers were obtained. After processing, the nucleic acid-removed decellularized adipose tissue fibers were squeezed dry using a juicer to obtain squeezed nucleic acid-removed decellularized adipose tissue fibers. Then, 10 times the mass of the squeezed nucleic acid-removed decellularized adipose tissue fibers (purified water at 25℃) were added to the squeezed nucleic acid-removed decellularized adipose tissue fibers and stirred and washed at 60 rpm for 5 minutes. The fibers were squeezed dry again using a juicer. This washing and squeezing process (i.e., repeated washing process) was repeated 5 times to obtain the washed nucleic acid-removed decellularized adipose tissue fibers. Then, they were placed in sterile bags and stored at 4℃. Nucleic acid-free decellularized adipose tissue fiber samples were homogenized using a high-shear homogenizer at 15000 rpm for 20 min to obtain a homogenate. The amount of pepsin was calculated based on a homogenate-to-pepsin mass ratio of 100:4 (pepsin activity concentration 3000 U / mg), and the pepsin was added to the homogenate and mixed thoroughly. The mixture was then incubated at 4°C with a shaking incubator for 72 h to obtain a pepsin-hydrolyzed sample. After hydrolysis, the pH of the pepsin-hydrolyzed sample was adjusted to 7.2 using a 0.1 mol / L sodium hydroxide aqueous solution and allowed to stand for 30 min. Finally, the sample was centrifuged at 8000 rpm for 20 min using a refrigerated centrifuge, the supernatant was discarded, and the precipitate was collected to obtain the precipitate.

[0085] Example 12 Example 12 is basically the same as Example 1, except that: ⑤ Dissolve the precipitate obtained in step ④ in a 0.01 mol / L hydrochloric acid solution (acid solution) to obtain dialysate. The mass ratio of precipitate to hydrochloric acid solution is 1:25. Then, place the dialysate into a dialysis bag with a molecular weight cutoff of 50 kDa, clamp both ends with clamps, and place it in a dialysis tank for gradient dialysis. The dialysis is performed using purified water as the dialysate for three days. During dialysis, the mass ratio of dialysate to dialysate is 1:10, and the dialysate is changed twice a day. The temperature of the dialysate and dialysate is maintained at 4°C during dialysis. The resulting solution was then placed in a stainless steel freeze-drying tray, which was then covered with a medical packaging bag, sealed, and placed in a freeze dryer for freeze-drying. The resulting freeze-dried product was adipose-derived collagen. The freeze-drying process involved first cooling from room temperature to -45°C (uniform cooling rate, cooling time 1 hour) and pre-freezing at -45°C for 3 hours, then heating to -10°C (uniform heating rate, heating time 1 hour) and freeze-drying at -10°C for 48 hours, and finally heating to 10°C and drying at 10°C for 10 hours. The entire freeze-drying process was carried out under an absolute pressure of 200 μbar.

[0086] Example 13 Example 13 is basically the same as Example 1, except that: ⑤ Dissolve the precipitate obtained in step ④ in a 0.01 mol / L hydrochloric acid solution (acid solution) to obtain dialysate. The mass ratio of precipitate to hydrochloric acid solution is 1:25. Then, put the dialysate into a dialysis bag with a molecular weight cutoff of 50 kDa, clamp both ends with clamps, and place it in a dialysis tank for gradient dialysis. The dialysis is performed for three days using hydrochloric acid solution with a pH of 3 as the dialysis water. During dialysis, the mass ratio of dialysate to dialysis water is 1:10, and the dialysis water is changed twice a day. During dialysis, the temperature of the dialysate and dialysis water is maintained at 4°C. The solution obtained after dialysis was placed in a stainless steel freeze-drying tray, which was then covered with a medical packaging bag, sealed, and placed in a freeze dryer for freeze-drying. The freeze-dried product was adipose-derived collagen. The freeze-drying process involved first cooling from room temperature to -45°C (uniform cooling rate, cooling time 1 hour) and pre-freezing at -45°C for 3 hours, then heating to -10°C (uniform heating rate, heating time 1 hour) and freeze-drying at -10°C for 48 hours, and finally heating to 10°C and drying at 10°C for 10 hours. The entire freeze-drying process was carried out under an absolute pressure of 200 μbar.

[0087] Example 14 Example 14 is basically the same as Example 1, except that: ⑤ Dissolve the precipitate obtained in step ④ in a 0.01 mol / L hydrochloric acid solution (acid solution) to obtain dialysate. The mass ratio of precipitate to hydrochloric acid solution is 1:25. Then, put the dialysate into a dialysis bag with a molecular weight cutoff of 50 kDa, clamp both ends with clamps, and place it in a dialysis tank for gradient dialysis. The dialysis is performed using hydrochloric acid solution with a pH of 5 as the dialysate for three days. During dialysis, the mass ratio of dialysate to dialysate is 1:10, and the dialysate is changed twice a day. The temperature of the dialysate and dialysate is maintained at 4°C during dialysis. The solution obtained after dialysis was placed in a stainless steel freeze-drying tray, which was then covered with a medical packaging bag, sealed, and placed in a freeze dryer for freeze-drying. The freeze-dried product was the adipose-derived collagen. The freeze-drying process involved first cooling from room temperature to -45°C (uniform cooling rate, cooling time 1 hour) and pre-freezing at -45°C for 3 hours, then heating to -10°C (uniform heating rate, heating time 1 hour) and freeze-drying at -10°C for 48 hours, and finally heating to 10°C and drying at 10°C for 10 hours. The entire freeze-drying process was carried out under an absolute pressure of 200 μbar.

[0088] Example 15 Example 15 is basically the same as Example 1, except that: ① Fresh pork fat tissue was washed with purified water and crushed into small particles with a diameter of 3 mm using a high-speed homogenizer to obtain the material. The material was frozen at -80℃ for 24 hours, and then completely thawed in a 37℃ water bath. This freeze-thaw cycle was repeated 3 times. After the freeze-thaw cycle, the material was homogenized using a high-shear homogenizer (15000 rpm, 10 min). The homogenate was then centrifuged at 8000 rpm for 30 min at 4℃. The upper fat layer was discarded, and the precipitate was collected. The precipitate was then washed twice with purified water at 4℃ (each time with 5 times the mass of purified water, stirred for 5 min, centrifuged, and the supernatant was discarded) to obtain the preliminarily defatted material.

[0089] The fat content of the adipose-derived collagen prepared in this embodiment was measured to be 9.5%, the bFGF content was 28.6 ng / g (dry weight), the VEGF content was 62.3 ng / g (dry weight), the relative percentage of type IV collagen was 0.42%, and the relative percentage of type VI collagen was 0.51%.

[0090] Comparative Example 1 Comparative Example 1: A collagen porous microsphere was prepared according to Example 1 of CN117482292A.

[0091] Comparative Example 2 Comparative Example 2: A collagen microsphere was prepared according to Example 1 of CN121801122A.

[0092] The porosity, pore connectivity, and compressive modulus of the porous collagen microspheres prepared in Examples 1-7 and the microspheres finally prepared in Comparative Examples 1-2 were tested, and the results are shown in Table 2. The pore connectivity of the internal porous structure of the porous collagen microspheres was observed by focused ion beam scanning electron microscopy, the porosity was measured by liquid displacement method, and the compressive modulus was measured by atomic force microscopy indentation method (n=10).

[0093] Table 2 In Table 2, the symbol " / " indicates that the performance metric was not tested.

[0094] The cell adhesion rates of the porous collagen microspheres finally prepared in Examples 1-15 and the microspheres finally prepared in Comparative Examples 1-2 were measured, and the results are shown in Table 3. The cell adhesion rate was determined by sterilizing the microspheres with ethylene oxide and then reacting them with L929 mouse fibroblasts (1×10⁻⁶ cells). 5 The cells were co-cultured with microspheres (cells / mL) for 4 h, and then washed with PBS buffer to remove unadhered cells. The number of cells adhering to the microspheres was determined by the CCK-8 assay, and the cell adhesion rate was calculated based on the initial seeding number (n=3).

[0095] Table 3

[0096] As shown in Tables 2 and 3, the porous collagen microspheres prepared in the preferred embodiment of the present invention have a cell adhesion rate of over 84% and a compressive modulus of over 120 kPa. The higher cell adhesion rate is conducive to the rapid adhesion and proliferation of cells on the surface of the microspheres, thus providing a cellular basis for subsequent vascularization. At the same time, the higher compressive modulus can provide sufficient mechanical support to resist tissue contraction and external pressure after implantation, maintain a stable three-dimensional porous structure to guide blood vessel ingrowth. Thus, the porous collagen microspheres prepared in the preferred embodiment of the present invention can promote early blood supply establishment by enhancing the interaction between cells and scaffolds, and can also ensure the stable maturation of the neovascular network by utilizing mechanical adaptability, thereby improving the survival rate of fat grafts.

[0097] Example 16 This embodiment provides a method for preparing dual growth factor sustained-release microspheres with a core-shell structure. The porous collagen microspheres used are porous collagen microspheres encapsulated with PDGF-BB (platelet-derived growth factor). The preparation of porous collagen microspheres encapsulated with PDGF-BB is basically the same as that of porous collagen microspheres in Example 1, except that step ⑥ is: first, the amino acids are dissolved in a 0.01 mol / L hydrochloric acid solution, and after complete dissolution, the adipose-derived collagen and PDGF obtained in step ⑤ are added. PDGF-BB (platelet-derived growth factor) was dissolved and homogenized by stirring at 4°C to obtain a collagen / amino acid mixture solution with added PDGF-BB. The collagen concentration in the collagen / amino acid mixture solution with added PDGF-BB was 2 w / v, and the mass of amino acids was 10% of the dry weight of decellularized adipose collagen. The amino acids consisted of glycine, proline, and hydroxyproline in a mass ratio of 2:1:1. The concentration of PDGF-BB in the collagen / amino acid mixture solution with added PDGF-BB was 25 μg / mL.

[0098] The preparation method of the core-shell structured dual growth factor sustained-release microspheres includes the following steps: S1. Perform the same steps ① to ④ as in Example 1, and then proceed to step ⑤. Step ⑤ involves dissolving the precipitate obtained in step ④ in a 0.01 mol / L hydrochloric acid solution (acid solution) to obtain a clear dialysate. The mass ratio of precipitate to hydrochloric acid solution is 1:25. The dialysate is then placed into a dialysis bag with a molecular weight cutoff of 50 kDa, clamped at both ends, and placed in a dialysis tank for gradient dialysis. The gradient dialysis involves first using a hydrochloric acid solution with a pH of 3.0 as the dialysate for one day, then using a hydrochloric acid solution with a pH of 5.0 as the dialysate for one day, and finally using purified water as the dialysate. The collagen solution was obtained by dialysis with dialysate for one day. During dialysis, the mass ratio of dialysate to dialysate was 1:10, and the dialysate was changed twice daily. The temperature of both the dialysate and dialysate was maintained at 4°C during dialysis. The collagen solution obtained after gradient dialysis was diluted with pre-cooled (pre-cooled to 4°C) MES buffer to a collagen concentration of 1 mg / mL, with the entire process performed at 4°C. The pH of the system was then adjusted to 5.5 to obtain the collagen MES buffer. 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC hydrochloride) and N-hydroxyl... Succinimide (NHS) was prepared into 400 mM EDC hydrochloride stock solution and 100 mM NHS stock solution. The NHS stock solution and EDC hydrochloride stock solution were added sequentially to the MES buffer of collagen to achieve a final NHS concentration of 5 mM and a final EDC hydrochloride concentration of 20 mM, and then mixed thoroughly to obtain the cross-linking reaction system. Vascular endothelial growth factor (VEGF) was dissolved in MES buffer pre-cooled to 4°C (VEGF to MES buffer mass ratio of 1:1.5), and added to the cross-linking reaction system within 10 minutes to achieve a final VEGF concentration of 5 μg / mL. The mixture was then incubated at 4°C. The reaction mixture was incubated at 4°C for 4 hours (shaking at low speed (50 rpm) on a shaker); then, a 0.1 M Na2HPO4 aqueous solution was added, with the volume being 1 / 10 of the total volume of the crosslinking reaction system. The mixture was incubated at 4°C for 3 hours to quench unreacted EDC hydrochloride and / or NHS and to hydrolyze unstable intermediates, yielding a reaction mixture. The reaction mixture was then ultrafiltered through an ultrafiltration membrane to remove byproducts and ungrafted VEGF, and concentrated through the concentration system of the ultrafiltration equipment to a VEGF-grafted collagen mass-volume concentration of 0.8 w / v%, yielding a VEGF-grafted collagen solution.

[0099] S2. The porous collagen microspheres coated with PDGF-BB were suspended at a concentration of 5 mg / mL in PBS buffer at pH 7.2 as the inner phase (also referred to as the core suspension); the collagen solution grafted with VEGF was used as the intermediate phase (shell precursor solution); mineral oil containing 1 wt% Span 80 (i.e., mineral oil with added Span 80) was used. The oil phase (concentration of 80% at 1 wt%) was used as the external phase. A coaxial microfluidic chip was used, with three inlets connected to the internal phase, intermediate phase, and external phase respectively. The flow rates were controlled at 5 μL / min for the internal phase, 10 μL / min for the intermediate phase, and 250 μL / min for the external phase, generating stable core-shell W / O / W droplets in the flow focusing region. The core-shell W / O / W droplets were collected in mineral oil containing 0.2 wt% glutaraldehyde (i.e., the mineral oil contained 0.2 wt% glutaraldehyde) for cross-linking and curing (cross-linking and curing at room temperature 25℃ for 3 h) to obtain core-shell microspheres. The core-shell microspheres were centrifuged (1500 rpm, 5 min) to remove the oil phase, washed twice with n-hexane, and then washed three times with PBS buffer at pH 7.2. The cleaned microspheres are freeze-dried to obtain dual growth factor sustained-release microspheres with a core-shell structure. In step S2, the freeze-drying process involves first cooling from room temperature to -30°C (uniform cooling rate, cooling time 1 h) and pre-freezing at -30°C for 3 h, then heating to -10°C (uniform heating rate, heating time 1 h) and drying at -10°C for 40 h (absolute pressure during drying is 0.2 mbar), then heating to 4°C (uniform heating rate, heating time 1 h) and drying at 4°C for 10 h (absolute pressure during drying is 0.1 mbar), and then heating to 25°C (uniform heating rate, heating time 1 h) and drying at 25°C for 3 h (absolute pressure during drying is 0.001 mbar).

[0100] In this embodiment, the prepared dual growth factor sustained-release microspheres with core-shell structure are combined with adipocytes to form an adipocyte-microsphere complex: Fresh porcine adipose tissue is washed with purified water, broken into small particles with a particle size of 3 mm using a high-speed homogenizer, and 0.9 w / v% physiological saline (the amount of physiological saline is 3 times the volume of adipose tissue) is added to the broken adipose tissue. After mixing evenly, the mixture is centrifuged at 1200 rpm for 2 minutes, the upper layer of fat and the lower layer of blood are discarded, and the middle layer of purified fat is collected. Add 0.9 w / v% physiological saline to the collected purified fat for washing (the volume of 0.9 w / v% physiological saline is 5 times the volume of purified fat; washing is done by stirring at 50 rpm for 1 min), followed by centrifugation (1200 rpm for 2 min). Discard the supernatant. Repeat the washing and centrifugation process twice to obtain treated adipocytes. Mix the treated adipocytes with the core-shell structured dual growth factor sustained-release microspheres to obtain the fat-microsphere complex. The amount of treated adipocytes is 5% of the mass of the core-shell structured dual growth factor sustained-release microspheres.

[0101] Example 17 Example 17 is basically the same as Example 16, except that the porous collagen microspheres used are porous collagen microspheres coated with PDGF-BB (platelet-derived growth factor). The preparation of the porous collagen microspheres coated with PDGF-BB (platelet-derived growth factor) is basically the same as that in Example 2, except that step ⑥ is: first, the amino acids are dissolved in a 0.01 mol / L hydrochloric acid solution, and after complete dissolution, the adipose-derived collagen and PDGF-BB (platelet-derived growth factor) obtained in step ⑤ are added. Platelet-derived growth factor (PDGF-BB) was stirred and dissolved uniformly at a low temperature of 4°C to obtain a collagen / amino acid mixed solution with PDGF-BB. The collagen concentration in the collagen / amino acid mixed solution with PDGF-BB was 2 w / v, and the mass amount of amino acids was 10% of the dry weight of decellularized adipose collagen. The amino acids were composed of glycine, hydroxyproline and glutamine in a mass ratio of 2:1:1. The concentration of PDGF-BB in the collagen / amino acid mixed solution with PDGF-BB was 25 μg / mL.

[0102] The sustained-release performance of the core-shell structured dual growth factor sustained-release microspheres prepared in Examples 16 and 17 was tested, and the results are shown in Table 4. The cumulative release rate of VEGF and PDGF-BB was determined as follows: Using enzyme-linked immunosorbent assay (ELISA), the core-shell structured dual growth factor sustained-release microspheres were placed in PBS buffer (pH=7.2, 37℃), and the release media were collected at predetermined time points (24h, 14d, 28d). The contents of VEGF and PDGF-BB were detected using a specific ELISA kit, and the cumulative release rate was calculated. Cumulative release rate (%) = (cumulative release amount / total loading in microspheres) × 100%.

[0103] Table 4

[0104] As shown in Table 4, the cumulative release rates of VEGF in Examples 16 and 17 were 18.7% and 19.3% at 24 hours, respectively, exhibiting a rapid release characteristic in the early stage, which is beneficial for inducing vascular endothelial cell budding. The cumulative release rates at 14 days reached 78.5% and 76.7%, respectively, and at 28 days, they reached 86.6% and 84.9%, respectively, indicating that VEGF was basically completely released within two weeks. The cumulative release rates of PDGF-BB at 28 days were 65.3% and 63.8%, respectively, with approximately 35% still unreleased, exhibiting a long-term sustained-release characteristic. The sequential release characteristics of rapid VEGF release and sustained PDGF-BB release mimic the physiological process of angiogenesis, which is beneficial for promoting the establishment of functional blood supply around the fat graft.

[0105] The present invention also provides the performance test results of the fat-microsphere complexes finally obtained in Examples 16-17, as shown in Table 5. The cell viability test involved culturing the fat-microsphere complex in vitro for 28 days, followed by Calcein-AM / PI dual-fluorescence staining. Live cells were stained with Calcein-AM (green fluorescence), and dead cells were stained with PI (red fluorescence). Five fields of view were randomly selected under a fluorescence microscope for observation and counting. The fat cell viability (%) was calculated as: (number of live cells / (number of live cells + number of dead cells)) × 100%. The vascular density measurement involved implanting the fat-microsphere complex into an animal (subcutaneous in nude mice). After 28 days, the graft and surrounding tissue were completely removed, paraffin sections were prepared, and CD31 immunohistochemical staining (CD31 is a marker of vascular endothelial cells) was performed. Five high-power fields of view were randomly selected under an optical microscope inside the graft and at the graft-host interface to count the number of CD31-positive blood vessels. The average value was calculated, with units of roots / mm. 2 .

[0106] Table 5 The parts of this invention not described in detail are techniques known to those skilled in the art.

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing porous collagen microspheres, characterized in that, The method includes the following steps: (1) Mix collagen and amino acids evenly with hydrochloric acid solution to obtain collagen / amino acid mixed solution; (2) The collagen / amino acid mixture solution was granulated by liquid nitrogen spraying to obtain frozen microspheres; (3) The frozen microspheres were subjected to first freeze-drying, cross-linking and second freeze-drying in sequence to prepare porous collagen microspheres.

2. The preparation method according to claim 1, characterized in that: The concentration of the hydrochloric acid solution is 0.005~0.015 mol / L; The collagen / amino acid mixture contains collagen at a concentration of 1-3 w / v%. The mass percentage of the amino acids is 5-15% of the mass percentage of the collagen, preferably 10%.

3. The preparation method according to claim 1, characterized in that: The amino acid is composed of glycine, proline, and hydroxyproline in a mass ratio of 2:(0.5~1.5):(0.5~1.5); or The amino acid is composed of glycine, hydroxyproline and glutamine in a mass ratio of 2:(0.5~1.5):(0.5~1.5).

4. The preparation method according to claim 1, characterized in that: The nozzle used for liquid nitrogen spray granulation has a diameter of 0.5~2.0cm and a spray pressure of 0.2~0.4bar.

5. The preparation method according to claim 1, characterized in that: The crosslinking agents used are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide; The crosslinking process involves placing the microspheres obtained after the first freeze-drying into a crosslinking agent solution for crosslinking. The crosslinking agent solution contains 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and an ethanol solution. Preferably, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the crosslinking agent solution is 20~40 mmol / L; Preferably, the crosslinking agent solution contains 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide in a mass ratio of 1:(3~6). Preferably, the ethanol solution is composed of anhydrous ethanol and water in a volume ratio of (6~8):(2~4).

6. The preparation method according to any one of claims 1 to 5, characterized in that, The collagen is adipose-derived collagen, and the preparation of the adipose-derived collagen includes the following steps: (a) After cleaning the adipose tissue, it is broken into small particles to obtain the material. Then, isopropanol is used to perform a gradient degreasing treatment on the material with an increasing material-to-liquid ratio to obtain the preliminarily degreased material. (b) The preliminarily defatted material was decellularized using a phosphate buffer solution containing trypsin and disodium EDTA to obtain decellularized material; (c) Isopropanol was used to perform a secondary degreasing treatment on the decellularized material to obtain decellularized adipose tissue fibers; (d) First, DNA and RNA enzymes were used to treat the decellularized adipose tissue fibers to remove nucleic acid from the decellularized adipose tissue fibers. Then, the nucleic acid was soaked in acid soaking solution to remove the decellularized adipose tissue fibers and then homogenized to obtain a homogenate. Then, pepsin was used to enzymatically hydrolyze the homogenate and then neutralized. Finally, the precipitate was obtained by centrifugation. (e) The precipitate is dissolved in an acidic solution and subjected to gradient dialysis with increasing pH value. Finally, it is freeze-dried to obtain the freeze-dried product, which is the decellularized fat collagen.

7. The preparation method according to claim 6, characterized in that: The adipose-derived collagen protein comprises type I collagen, type IV collagen, type V collagen, and type VI collagen. Type I collagen accounts for more than 90% of the protein mass of the adipose-derived collagen protein, type IV collagen accounts for 1.0-1.5% of the protein mass of the adipose-derived collagen protein, type V collagen accounts for 3-3.5% of the protein mass of the adipose-derived collagen protein, and type VI collagen accounts for 1.5-2.0% of the protein mass of the adipose-derived collagen protein. The residual DNA content is less than 50 ng / mg. Preferably, based on the dry weight of the adipose-derived collagen, the adipose-derived collagen contains ≥100 ng / g of vascular endothelial growth factor, ≥30 ng / g of basic fibroblast growth factor, ≥8 ng / g of transforming growth factor-β1, and ≥1% by mass of glycosaminoglycans.

8. The preparation method according to claim 6, characterized in that: In step (a): the gradient degreasing process involves first mixing the material with isopropanol at a material-to-liquid ratio of 1:(1~5), preferably 1:(4~5), stirring at 37°C for 5~15 min, and then centrifuging once. Then, the material obtained from the first centrifugation is mixed with isopropanol at a material-to-liquid ratio of 1:(1~3), preferably 1:(2~3), stirring at 37°C for 15~25 min, and then centrifuging a second time. Finally, the material obtained from the second centrifugation is mixed with isopropanol at a material-to-liquid ratio of 1:(1~2), preferably 1:(1~1.5), stirring at 37°C for 25~35 min, and then centrifuging a third time. In step (d): the pH of the acid soaking solution is 1.8~2.2, the acid in the acid soaking solution is HCl, and the acid soaking solution also contains 3~8wt% ethanol. During soaking, the dry weight of the nucleic acid removed from decellularized adipose tissue fibers and the mass ratio of the acid soaking solution are (3~6) mg:1g. The soaking is carried out at 3~10℃, preferably 4℃, for 1~2 hours. The homogenization speed is 10000~20000rpm, preferably 15000rpm, for 10~30min. When using pepsin for enzymatic hydrolysis, the enzyme activity concentration of the pepsin is 2000~4000U / mg, preferably 3000U / mg. The mass ratio of the homogenate to the pepsin is 100:(1~10). The enzymatic hydrolysis is carried out at 3~10℃, preferably 4℃, for 60~96 hours. The neutralization is carried out to a pH of 7.0~7.2; and / or In step (e): the precipitate is dissolved in an acid solution to obtain dialysate, and then the dialysate is placed in a dialysis bag with a molecular weight cutoff of 35~100kDa for gradient dialysis. The gradient dialysis is performed by first dialysis with hydrochloric acid solution with pH 2.8~3.2 as dialysate for one day, then dialysis with hydrochloric acid solution with pH 4.8~5.2 as dialysate for one day, and finally dialysis with water as dialysate for one day. Preferably, the mass ratio of dialysate to dialysate is 1:(10~20). Preferably, the temperature of dialysate and dialysate is maintained at 4~10°C during dialysis.

9. Porous collagen microspheres prepared by any one of claims 1 to 8.

10. The application of porous collagen microspheres prepared by any one of claims 1 to 8 as the core material of tissue engineering scaffolds, cell culture carriers, drug delivery carriers, or core-shell structured microspheres; preferably, the core-shell structured microspheres are dual growth factor sustained-release microspheres with a core-shell structure; preferably, the dual growth factors are platelet-derived growth factor PDGF-BB and vascular endothelial growth factor VEGF.

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