Animal decellularized omentum particle loaded decellularized matrix hydrogel as well as preparation method and application thereof

Abstract

The invention provides acellular matrix hydrogel loaded with animal acellular omentum particles as well as a preparation method and application of the acellular matrix hydrogel loaded with the animal acellular omentum particles, and relates to the technical field of tissue engineering and regenerative medicine. Comprising a decellularization treatment agent, a decellularization matrix, functional particles, an enzymolysis agent, a cross-linking stabilizer, a functional additive and a solvent, the functional additive is prepared from PDA coated Fe3O4 magnetic nano particles, phosphorylated polyglycerol sebacate and liposome coated silver nano particles. The PDA-Fe3O4 magnetic nanoparticles of the functional additive generate mechanical signals under the action of an external magnetic field, a Piezo1 mechanical gating cation channel is activated to promote calcium ion internal flow, and the autonomous mineralization ability of phosphorylated polyglycerol sebacate is combined, so that the activity of the bone differentiation marker alkaline phosphatase is effectively improved, and the bone differentiation marker alkaline phosphatase is prepared. The combination of the PDA coated Fe3O4 magnetic nanoparticles and the phosphorylated polyglycerol sebacate integrates a magnetic response mineralization system.

Description

Technical Field

[0001] This invention relates to the fields of tissue engineering and regenerative medicine, and more specifically, to a decellularized matrix hydrogel loaded with decellularized animal reticulum particles, its preparation method, and its application. Background Technology

[0002] Decellularized matrix hydrogels loaded with decellularized omentum particles are a type of functional composite biomaterial that uses natural animal tissues such as the greater omentum of livestock such as pigs and cattle as the core raw material. Through decellularization treatment, the natural structure and bioactivity of the extracellular matrix (ECM) are preserved, and decellularized omentum particles are combined to form a functional composite biomaterial. It has the advantages of injectability, biocompatibility and the ability to regulate tissue repair, and has now become an important research direction in the fields of tissue engineering and regenerative medicine.

[0003] Although decellularized matrix hydrogels loaded with decellularized animal reticulum particles have shown broad application prospects, the insufficient bioactivity of decellularized matrix hydrogels prepared by related technologies and the contradiction between antibacterial properties and biocompatibility restrict the development of the technology. Simple decellularized matrix is ​​difficult to provide sufficient osteogenic differentiation signals, while the addition of high concentrations of antibacterial agents will significantly reduce cell activity, leading to a cytotoxicity level of 2 or higher. Summary of the Invention

[0004] This invention aims to address the problems of insufficient bioactivity of decellularized matrix hydrogels, the contradiction between antibacterial properties and biocompatibility, and the difficulty in providing sufficient osteogenic differentiation signals.

[0005] To address the above problems, this invention provides a decellularized matrix hydrogel loaded with decellularized animal reticulum particles, its preparation method, and its application.

[0006] In a first aspect, the present invention provides a decellularized matrix hydrogel loaded with decellularized animal membrane particles, comprising a decellularization treatment agent, a decellularized matrix, functional particles, an enzymatic hydrolysant, a crosslinking stabilizer, a functional additive, and a solvent; The functional additives include PDA@Fe3O4 magnetic nanoparticles, phosphorylated polyglycerol sebacic acid esters, and liposome-encapsulated silver nanoparticles.

[0007] Optionally, the decellularization treatment agent includes an amphoteric detergent, deoxyribonuclease I, and ribonuclease A.

[0008] Optionally, the decellularized matrix is ​​a decellularized product of porcine or bovine omentum.

[0009] Optionally, the functional particles include decellularized membrane particles, hyaluronic acid, and nanocellulose, wherein the decellularized membrane particles are derivative products obtained by freeze-drying and grinding a partially decellularized matrix.

[0010] Optionally, the decellularized membrane particles have a particle diameter of less than 50 μm; the hyaluronic acid has a molecular weight of 700 kDa to 900 kDa; and the nanocellulose is eucalyptus pulp (2,2,6,6-tetramethylpiperidine-1-oxygen radical) oxidized nanocellulose or bacterial nanocellulose.

[0011] Alternatively, the enzymatic hydrolysant may include pancreatic enzymes, collagenases, and matrix lysins.

[0012] Alternatively, cross-linking stabilizers include gardenia extract and chitosan.

[0013] Secondly, the present invention provides a method for preparing a decellularized matrix hydrogel loaded with decellularized animal reticulum particles as described above, comprising the following steps: S1: Select animal reticulum tissue, cut it into small pieces, add decellularization agent and solvent, and treat it at a constant temperature of 36 to 38°C with shaking. Then rinse it with solvent buffer to obtain decellularized matrix. S2: Take a portion of the decellularized matrix obtained in step S1 and immerse it in liquid nitrogen. After taking it out, freeze-mill it and sieve it to obtain decellularized membrane particles. Add hyaluronic acid solution and use ultrasonic treatment to modify the surface of the decellularized membrane particles. Add nanocellulose suspension and use ultrasonic treatment to form functional particle suspension. S3: Take the decellularized matrix obtained in the remaining step S1, add the enzymatic hydrolysant and mix, add solvent to adjust the solid-liquid ratio to 1:15 to 25, enzymatically hydrolyze in a constant temperature water bath at 36 to 38°C, monitor in real time with a dynamic rheometer, and terminate the enzymatic hydrolysis when the storage modulus is not lower than 1.5 kPa. Add the functional particle suspension and cross-linking stabilizer obtained in step S2 in sequence, add the functional additive after magnetic stirring, and obtain the hydrogel precursor solution after vacuum degassing. S4: Transfer the hydrogel precursor solution obtained in step S3 into a mold, and let it stand in a constant temperature incubator at 36 to 38°C to complete the initial gelation. Then place it in a magnetic field to promote the orderly arrangement of magnetic particles and form a decellularized matrix hydrogel with magnetic response characteristics loaded with decellularized animal membrane particles.

[0014] Optionally, the decellularization treatment agent includes an amphoteric detergent, deoxyribonuclease I, and ribonuclease A. Step S1, which involves adding the decellularization treatment agent and solvent and treating with constant temperature shaking at 36 to 38°C, includes: adding a mixture of the amphoteric detergent and solvent and treating with constant temperature shaking at 36 to 38°C; subsequently adding a mixed solution of deoxyribonuclease I and ribonuclease A and continuing to treat with shaking at 36 to 38°C.

[0015] Thirdly, the present invention provides an application of the decellularized matrix hydrogel loaded with decellularized animal membrane particles as described above in the field of injectable functional composite biomaterials or implantable functional composite biomaterials.

[0016] The beneficial effects of the decellularized matrix hydrogel loaded with decellularized animal reticulum particles, its preparation method, and its application of the present invention are as follows: The PDA@Fe3O4 magnetic nanoparticles, composed of polydopamine (PDA) encapsulating a Fe3O4 magnetic core, generate mechanical signals such as magnetostrictive vibration or directional rotation under the action of an external magnetic field. These signals are transmitted to the cell membrane through direct contact conduction or matrix tension transmission. When the mechanical signals are transmitted to the cell membrane, they mainly activate the Piezo1 mechanically gated cation channels through changes in membrane curvature and lipid bilayer tension conduction. After the Piezo1 channels open, calcium ions rapidly flow inward through the central pore, upregulating RUNX2 and OCN through calcium signaling. It contains osteogenic genes to promote bone defect repair. Combined with the autonomous mineralization ability of phosphorylated polyglycerol sebacic acid (PGS-P), it effectively enhances the activity of alkaline phosphatase (ALP), a marker of bone differentiation. The combination of PDA@Fe3O4 magnetic nanoparticles and phosphorylated polyglycerol sebacic acid integrates a magnetically responsive mineralization system. Liposomes encapsulate silver nanoparticles, and the liposome encapsulation technology enables the sustained release control of silver nanoparticles, effectively maintaining the antibacterial rate while reducing the cytotoxicity level to below grade 1, effectively solving the contradiction between antibacterial and biocompatibility. Attached Figure Description

[0017] Figure 1 This is a flowchart illustrating the preparation method of a decellularized matrix hydrogel loaded with decellularized animal reticulum particles, according to an embodiment of the present invention. Detailed Implementation

[0018] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Although some embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the present invention. It should be understood that the accompanying drawings and embodiments of the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0019] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention's description is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "comprising" and its variations as used herein are open-ended, meaning "including but not limited to"; the term "based on" means "at least partially based on"; the term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment"; the term "some embodiments" means "at least some embodiments"; and the term "optionally" means "optional embodiments". Definitions of other terms will be given in the following description. It should be noted that the concepts of "first," "second," etc., mentioned in this invention are used to distinguish different objects, not to describe a specific order or hierarchy. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, unless otherwise stated, "a plurality of" means two or more.

[0020] In related technologies, poor batch-to-batch consistency of decellularized matrix hydrogels is a key issue restricting their clinical application. This is mainly due to individual differences in animal raw materials and instability in the decellularization process, resulting in fluctuations of 15-20% in key components such as collagen and glycosaminoglycans, directly affecting the mechanical properties and bioactivity of the hydrogel. Traditional decellularization processes often use anionic detergents such as SDS, which can effectively remove cellular components but excessively damage the natural structure of the ECM, leading to a decrease in collagen retention rate to below 60% and a high level of residual DNA, increasing the risk of immune rejection. Insufficient particle dispersibility is another prominent problem. Decellularized membrane particles are prone to aggregation in the gel matrix, especially at concentrations exceeding 5 mg / mL, resulting in significant sedimentation and uneven mechanical properties within the hydrogel. The compressive modulus deviation can reach more than 20%, affecting the stability of the repair effect. Poor matching between mechanical properties and degradation rate is also a common problem. Traditional chemical cross-linking agents such as glutaraldehyde can improve mechanical strength, but they can prolong the degradation cycle to more than 12 weeks, which is incompatible with the needs of tissue regeneration. Meanwhile, hydrogels with low cross-linking degrees face the problem of insufficient mechanical support.

[0021] To address the problems existing in the aforementioned related technologies, embodiments of the present invention provide a decellularized matrix hydrogel loaded with decellularized animal reticulum particles, its preparation method, and its application.

[0022] An embodiment of the present invention provides a decellularized matrix hydrogel loaded with decellularized animal membrane particles, comprising a decellularization treatment agent, a decellularized matrix, functional particles, an enzymatic hydrolysant, a crosslinking stabilizer, a functional additive, and a solvent; The functional additives include PDA@Fe3O4 magnetic nanoparticles, phosphorylated polyglycerol sebacic acid esters, and liposome-encapsulated silver nanoparticles.

[0023] Specifically, decellularization agents refer to a combination of reagents used to remove cellular components while protecting the extracellular matrix. This can be achieved through the synergistic effect of amphoteric detergents and nucleases, effectively removing residual genetic material while maintaining the integrity of collagen fibers. Functional particles are composite components that enhance the functionality of materials. They can be constructed by combining homologous decellularized matrix derivatives with polysaccharides to improve batch stability and particle dispersibility. Enzymatic hydrolysants are combinations of enzymes that regulate the rate of matrix degradation. This can be achieved by adjusting the ratio of trypsin to collagenase, and controlling the gel formation process by dynamically monitoring the degree of enzymatic hydrolysis. Crosslinking stabilizers are natural substances that promote the formation of three-dimensional networks. These can be achieved using a composite system of plant extracts and chitosan, avoiding the cytotoxicity caused by chemical crosslinking. Functional additives are nanocomposite systems that endow materials with special functions. These can be achieved by combining magnetic nanoparticles with sustained-release antibacterial agents, simultaneously meeting the requirements for directional alignment and long-lasting antibacterial effects.

[0024] In this embodiment, the PDA@Fe3O4 magnetic nanoparticles of the functional additive consist of a Fe3O4 magnetic core encapsulated by polydopamine (PDA). Under the action of an external magnetic field, they generate mechanical signals such as magnetostrictive vibration or directional rotation, which are transmitted to the cell membrane through direct contact conduction or matrix tension conduction. When the mechanical signal is transmitted to the cell membrane, it mainly activates the Piezo1 mechanogated cation channel through changes in membrane curvature and lipid bilayer tension conduction. After the Piezo1 channel opens, calcium ions rapidly flow in through the central pore, upregulating RUNX2 and OCN through calcium signaling. It contains osteogenic genes to promote bone defect repair. Combined with the autonomous mineralization ability of phosphorylated polyglycerol sebacic acid (PGS-P), it effectively enhances the activity of alkaline phosphatase (ALP), a marker of bone differentiation. The combination of PDA@Fe3O4 magnetic nanoparticles and phosphorylated polyglycerol sebacic acid integrates a magnetically responsive mineralization system. Liposomes encapsulate silver nanoparticles, and the liposome encapsulation technology enables the sustained release control of silver nanoparticles, effectively maintaining the antibacterial rate while reducing the cytotoxicity level to below grade 1, effectively solving the contradiction between antibacterial and biocompatibility.

[0025] Optionally, the decellularized matrix hydrogel loaded with decellularized animal membrane particles, by weight, comprises 2.5-5 parts of decellularization treatment agent, 45-55 parts of decellularized matrix, 18-32 parts of functional particles, 1.9-3 parts of enzymatic hydrolysate, 4.2-6.4 parts of crosslinking stabilizer, 1.2-3.6 parts of functional additives, and 5-45 parts of solvent.

[0026] Optionally, the decellularization treatment agent includes an amphoteric detergent, deoxyribonuclease I, and ribonuclease A.

[0027] Specifically, amphoteric detergents are surfactants that possess both positive and negative charged groups. These can be achieved using 3-[(3-cholanamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS) or dodecyl dimethyl betaine (DDAB). They gently dissolve cell membrane structures through charge neutralization, reducing damage to collagen fibers. 3-[(3-cholanamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS) has the molecular formula C2... 32 H 58 N2O7S has a molecular weight of approximately 614.88. Deoxyribonuclease I is a hydrolase that specifically cleaves double-stranded DNA molecules. Specifically, it can be implemented using DNase I derived from bovine pancreas, which degrades residual genomic DNA by cleaving phosphodiester bonds. Ribonuclease A is an endonuclease that hydrolyzes single-stranded RNA molecules. Specifically, it can be implemented using RNase A derived from bovine pancreas, which removes residual RNA by cleaving the phosphodiester bonds between pyrimidine nucleotides.

[0028] In this optional embodiment, the amphoteric detergent preferentially acts on the cell membrane lipid bilayer, achieving cell lysis by reducing surface tension without disrupting the three-dimensional network structure of the extracellular matrix. By using an amphoteric detergent instead of traditional SDS, its critical micelle concentration ensures efficient decellularization while minimizing extracellular matrix (ECM) damage. Deoxyribonuclease I and ribonuclease A form a complementary mechanism of action; the former targets and degrades DNA fragments released from the cell nucleus, while the latter removes residual RNA components from the cytoplasm. During the treatment, after the amphoteric detergent completes the removal of cell debris, the two nucleases act on different nucleic acid types in stages, avoiding interference between enzyme activities while ensuring the thoroughness of nucleic acid degradation.

[0029] Optionally, by weight, the decellularization treatment agent includes 2-4 parts of amphoteric detergent, 0.3-0.6 parts of deoxyribonuclease I, and 0.2-0.4 parts of ribonuclease A.

[0030] Optionally, the decellularized matrix is ​​a decellularized product of porcine or bovine omentum.

[0031] Specifically, porcine omentum decellularized products refer to the extracellular matrix remaining after removing cellular components from porcine omentum tissue using decellularizing agents and enzymatic hydrolysants. Specifically, amphoteric detergents combined with nuclease treatment can be used to achieve complete removal of cellular components. Bovine omentum decellularized products refer to bovine omentum tissue treated using the same process, which retains the natural three-dimensional structure of collagen and glycosaminoglycans through a standardized decellularization procedure.

[0032] In this optional embodiment, pigs and cattle raised on a large scale have highly similar anatomical structures, and the extracellular matrix composition of their omentum tissue exhibits low individual variability within the species. By limiting the decellularized matrix to these two specific animals, it is possible to ensure that the collagen fiber arrangement and glycosaminoglycan content in the raw materials tend to be consistent. During the preparation process, after standardized decellularization treatment of the pig or bovine omentum, the residual DNA content can be controlled below 0.5 μg / mg, while maintaining a collagen retention rate of over 80%. The raw material screening standard established in this way effectively avoids the impact of differences in matrix composition between different species on the hydrogel performance. Standardized control of the decellularized matrix composition is achieved, significantly reducing batch-to-batch performance fluctuations caused by differences in raw material sources. The regular arrangement of collagen fibers and the stable distribution of glycosaminoglycans provide uniform mechanical support for the hydrogel, while ensuring the reproducibility of cell adhesion and proliferation behavior.

[0033] Optionally, the functional particles include decellularized membrane particles, hyaluronic acid, and nanocellulose, wherein the decellularized membrane particles are derivative products obtained by freeze-drying and grinding a partially decellularized matrix.

[0034] Specifically, decellularized reticulum particles refer to micron-sized particles formed by mechanically grinding a decellularized matrix after freezing it in liquid nitrogen. This can be achieved by immersing the particles in liquid nitrogen followed by low-temperature crushing using a ball mill. This process physically controls particle size and reduces surface charge heterogeneity. Hyaluronic acid refers to a high molecular weight hydrophilic polymer with a molecular weight of 700kDa to 900kDa. It can be achieved by ultrasonic-assisted dissolution to form an encapsulated solution, where long-chain molecules adsorb onto the particle surface to form a steric hindrance layer. Nanocellulose refers to nanoscale fiber materials produced from plant fibers through oxidation or bacterial fermentation. Specifically, it can be achieved by preparing a nanocellulose suspension from eucalyptus pulp using the TEMPO oxidation method with 2,2,6,6-tetramethylpiperidine-1-oxygen radicals. This suspension forms a three-dimensional support structure in the liquid phase through a nanofiber network.

[0035] In this optional embodiment, decellularized membrane particles are freeze-dried and ground to form uniform particles with controllable particle size, reducing the tendency to aggregate due to differences in surface roughness; hyaluronic acid solution is treated with ultrasound to encapsulate the particle surface with polymer chains, reducing interfacial energy and forming a stable hydration layer; nanocellulose suspension is dispersed by ultrasound to form an interlinked fiber network, constructing a physical barrier framework around the functional particles, introducing a dual dispersion mechanism of hyaluronic acid modification and nanocellulose network, hyaluronic acid is modified by hydroxylation to increase the hydrophilicity of the particles, and nanocellulose is treated by ultrasound to form a three-dimensional support network, significantly prolonging the particle suspension stability and effectively controlling deviation.

[0036] Optionally, by weight, the functional particles include 15-25 parts of decellularized membrane particles, 1-3 parts of hyaluronic acid, and 2-4 parts of nanocellulose.

[0037] Optionally, the decellularized membrane particles have a particle diameter of less than 50 μm; the hyaluronic acid has a molecular weight of 700 kDa to 900 kDa; and the nanocellulose is eucalyptus pulp (2,2,6,6-tetramethylpiperidine-1-oxygen radical) oxidized nanocellulose or bacterial nanocellulose.

[0038] Specifically, the particle diameter of decellularized membrane particles refers to the maximum lateral dimension of the solid microparticles formed after freeze-drying and grinding. This can be achieved by combining liquid nitrogen cryogenic crushing with ball milling and sieving. Smaller particle sizes help reduce particle settling rates. The molecular weight of hyaluronic acid refers to the relative mass range of its polymer chains. The molecular weight distribution can be controlled using enzymatic hydrolysis or ultrafiltration. A suitable molecular weight range can balance solution viscosity and processing performance. Nanocellulose refers to nanoscale cellulose materials prepared through chemical oxidation or microbial fermentation. Specifically, it can be produced using the TEMPO oxidation method to treat eucalyptus pulp or the static culture method of Acetobacter xylinum. Its surface charge characteristics or three-dimensional network structure can provide steric hindrance effects.

[0039] In this optional embodiment, limiting the particle diameter inhibits sedimentation by reducing the effect of gravity, selecting the molecular weight of hyaluronic acid maintains particle suspension by adjusting the rheological properties of the solution, and introducing nanocellulose prevents particle aggregation through electrostatic repulsion or physical barrier mechanisms. These three elements work synergistically to form a three-dimensional dispersion system of particle size control, rheological adjustment, and steric hindrance, achieving uniform particle distribution in the gel matrix without excessively increasing viscosity. By screening particle size, optimizing molecular weight, and selecting the type of nanocellulose, a multiple dispersion mechanism is constructed, significantly improving particle dispersion stability while maintaining processing fluidity. This solves the problem of uneven mechanical properties caused by insufficient dispersibility of decellularized membrane particles in the gel matrix, reduces particle aggregation, improves the uniformity of the internal structure of the hydrogel, and avoids the impact of excessively high solution viscosity on subsequent processing and molding.

[0040] Alternatively, the enzymatic hydrolysant may include pancreatic enzymes, collagenases, and matrix lysins.

[0041] Specifically, trypsin refers to a broad-spectrum protease capable of breaking down intercellular junction proteins, and can be produced using porcine pancreas extract. It promotes the release of cellular debris by cleaving intercellular junctions. Collagenase refers to an enzyme that specifically degrades collagen fibers, and can be produced using collagenase derived from Clostridium histolyticum. It releases embedded cellular components by cleaving collagen molecular chains. Matrix lysin refers to an enzyme preparation that regulates the activity of matrix metalloproteinases, and can be produced using recombinant human matrix lysin-1. It achieves controlled dissociation of the extracellular matrix by hydrolyzing specific proteoglycans.

[0042] In this optional embodiment, the three enzymes form a synergistic decellularization system under isothermal conditions. Trypsin preferentially acts on cell membrane junction proteins, causing the cell structure to loosen and disintegrate; collagenase directionally breaks down the collagen network encapsulating the cell, prompting cell fragments to detach from the matrix framework; matrix lysin selectively degrades specific proteoglycan components by regulating matrix metalloproteinase activity, preserving the integrity of glycosaminoglycans. This staged enzymatic hydrolysis mechanism avoids excessive erosion by chemical reagents, maintaining the three-dimensional topology of collagen fibers while removing DNA residues, providing a structural basis for the subsequent construction of bioactive hydrogels. Targeted removal of cellular components is achieved through the enzymatic hydrolysis system. In existing technologies, SDS treatment destroys the collagen cross-linking network, while the synergistic effect of collagenase and matrix lysin in this scheme can precisely control the degree of matrix dissociation, preserving the mechanical support function of the natural ECM. This effectively reduces structural damage to the extracellular matrix during decellularization, significantly reduces the immunogenicity caused by DNA residues, and maintains the bioactivity of collagen and glycosaminoglycans, providing a reliable guarantee for the preparation of hydrogel materials with stable biological functions.

[0043] Optionally, the enzymatic hydrolysate may include 0.8-1.2 parts pancreatic enzyme, 0.8-1.2 parts collagenase, and 0.3-0.6 parts matrix lysin by weight.

[0044] Alternatively, cross-linking stabilizers include gardenia extract and chitosan.

[0045] Specifically, gardenia extract refers to natural polyphenolic substances extracted from gardenia fruit. It can be obtained through a combination of water extraction and alcohol precipitation with macroporous resin purification. The geniposide it contains binds to collagen fibers through hydrogen bonds and hydrophobic interactions, promoting three-dimensional network cross-linking under mild conditions. Chitosan refers to cationic polysaccharides obtained by deacetylation of chitin. It can be obtained through copolymerization of D-glucosamine and N-acetyl-D-glucosamine linked by β-1,4-glycosidic bonds. Its amino groups bind to negatively charged extracellular matrix components through electrostatic interactions and form dynamically reversible Schiff base cross-linking points with the phenolic hydroxyl groups in gardenia extract.

[0046] In this optional embodiment, a synergistic crosslinking system of gardenia extract (i.e., genipin) and chitosan achieves precise control over mechanical properties and degradation rate. The polyphenols in the gardenia extract preferentially form non-covalent bonds with the hydroxyl and carboxyl groups of collagen fibers, constructing a primary crosslinking network. Chitosan forms secondary crosslinking support through electrostatic adsorption of sulfated glycosaminoglycans in the extracellular matrix via its amino groups. The biepoxy structure of genipin forms a stable heterocyclic structure with the amino groups of chitosan. Simultaneously, a controllable degradation cycle of 4-8 weeks is achieved by adjusting the degree of deacetylation of chitosan, matching the regeneration needs of different tissues. The formation of dynamic crosslinking points allows the hydrogel to dissipate energy through bond breaking and recombination under mechanical stress while maintaining overall structural integrity. The pH sensitivity of the Schiff base reaction causes the crosslinking network to gradually dissociate under physiological conditions, achieving synchronization between degradation rate and tissue regeneration rate. This method achieves a balanced regulation of the mechanical strength and degradation rate of hydrogels. While avoiding the cytotoxicity of synthetic crosslinking agents, it maintains structural stability through a dynamic crosslinking network. At the same time, it utilizes the antibacterial properties of natural components to ensure the biocompatibility of the material, thus solving the contradiction between excessively long degradation cycles and decreased biocompatibility in traditional chemical crosslinking systems.

[0047] like Figure 1 As shown, another embodiment of the present invention provides a method for preparing a decellularized matrix hydrogel loaded with decellularized animal reticulum particles as described above, comprising the following steps: S1: Select animal reticulum tissue, cut it into small pieces, add decellularization agent and solvent, and treat it at a constant temperature of 36 to 38°C with shaking. Then rinse it with solvent buffer to obtain decellularized matrix. S2: Take a portion of the decellularized matrix obtained in step S1 and immerse it in liquid nitrogen. After taking it out, freeze-mill it and sieve it to obtain decellularized membrane particles. Add hyaluronic acid solution and use ultrasonic treatment to modify the surface of the decellularized membrane particles. Add nanocellulose suspension and use ultrasonic treatment to form functional particle suspension. S3: Take the decellularized matrix obtained in the remaining step S1, add the enzymatic hydrolysant and mix, add solvent to adjust the solid-liquid ratio to 1:15 to 25, enzymatically hydrolyze in a constant temperature water bath at 36 to 38°C, monitor in real time with a dynamic rheometer, and terminate the enzymatic hydrolysis when the storage modulus is not lower than 1.5 kPa. Add the functional particle suspension and cross-linking stabilizer obtained in step S2 in sequence, add the functional additive after magnetic stirring, and obtain the hydrogel precursor solution after vacuum degassing. S4: Transfer the hydrogel precursor solution obtained in step S3 into a mold, and let it stand in a constant temperature incubator at 36 to 38°C to complete the initial gelation. Then place it in a magnetic field to promote the orderly arrangement of magnetic particles and form a decellularized matrix hydrogel with magnetic response characteristics loaded with decellularized animal membrane particles.

[0048] Specifically, isothermal oscillation treatment refers to using mechanical oscillation within a temperature range of 36-38℃ to ensure uniform penetration of the decellularization agent into the tissue. This can be achieved using a isothermal shaker at 120 rpm, a temperature range that maintains enzyme activity while preventing protein denaturation. Cryo-milling involves rapidly freezing the biological material with liquid nitrogen to embrittle it, followed by pulverization using a grinder. This can be achieved using a planetary ball mill at -196℃ and 300 rpm, a method that preserves the integrity of the ECM microstructure. Real-time monitoring using a dynamic rheometer involves continuously measuring the viscoelastic changes of the sample using a rotational rheometer. This can be achieved using a cone-plate measurement system scanning the storage modulus at a frequency of 1 Hz, a method that accurately determines the end point of enzymatic hydrolysis. Crosslinking stabilizers enhance network stability through the synergistic effect of physical entanglement and chemical bonding. This can be achieved by combining chitosan and gardenia extract in a 1:2 mass ratio; the resulting multi-layered crosslinked network can regulate the degradation rate. Magnetic field treatment refers to using a static magnetic field to induce the directional alignment of magnetic particles. Specifically, a 0.5T permanent magnet can be used to apply a magnetic field along the axial direction for 30 minutes. This treatment can construct anisotropic structures and improve mechanical uniformity.

[0049] In this optional embodiment, the processing parameters are precisely controlled by an automated oscillation system, which effectively controls fluctuations in collagen content and eliminates DNA residue to a large extent, significantly improving batch-to-batch consistency.

[0050] During the isothermal oscillation stage, damage to the ECM structure is reduced by controlling the processing temperature and time, while amphoteric detergents and nucleases are added stepwise to achieve gentle decellularization. The cryo-milling process rapidly freezes and fixes the ECM spatial conformation, combined with ultrasonic treatment to coat the particle surface with hyaluronic acid, creating steric hindrance. This, along with the three-dimensional network support of nanocellulose, inhibits particle sedimentation. Dynamic rheological monitoring during enzymatic hydrolysis captures the degree of collagen fiber decrosslinking; the reaction is terminated immediately when the storage modulus reaches a critical value, ensuring consistent network crosslinking density in each batch. The introduction of crosslinking stabilizers forms a composite network of reversible hydrogen bonds and stable covalent bonds, allowing for controlled degradation while maintaining mechanical strength. The magnetic field treatment stage regulates the orientation of magnetic particles to form an ordered channel structure, enhancing local mechanical support and providing directional guidance for cell migration.

[0051] The entire process is completed at low temperatures to maximize the preservation of growth factor activity. Compared to the random distribution of mechanical mixing, the oriented structure formed by magnetic field-induced alignment achieves a hydrogel compressive modulus anisotropy ratio of 3:1.

[0052] Stable retention of decellularized matrix components was achieved, resulting in a collagen retention rate of over 85%; a uniformly dispersed three-dimensional network structure was constructed, reducing the particle sedimentation rate to below 0.5% per hour; controllable degradation performance was obtained, with the degradation cycle precisely adjustable within the range of 4-8 weeks; an ordered magnetic arrangement structure was formed, increasing the hydrogel's compression resilience by 40%; it also possesses both antibacterial activity and cell compatibility, achieving a 99% inhibition rate against Staphylococcus aureus with a cell survival rate exceeding 95%.

[0053] Optionally, the decellularization treatment agent includes an amphoteric detergent, deoxyribonuclease I, and ribonuclease A. Step S1, which involves adding the decellularization treatment agent and solvent and treating with constant temperature shaking at 36 to 38°C, includes: adding a mixture of the amphoteric detergent and solvent and treating with constant temperature shaking at 36 to 38°C; subsequently adding a mixed solution of deoxyribonuclease I and ribonuclease A and continuing to treat with shaking at 36 to 38°C.

[0054] Specifically, amphoteric detergents are surfactants that possess both hydrophilic and hydrophobic groups. They can be implemented using dodecyl dimethyl betaine or cocamidopropyl betaine, whose molecular structure allows for the gentle removal of cell membrane debris without damaging the collagen fiber network of the extracellular matrix. Deoxyribonuclease I is a nuclease that specifically cleaves double-stranded DNA. It can be implemented using enzyme preparations derived from bovine pancreas, breaking the DNA strand by hydrolyzing phosphodiester bonds. Ribonuclease A is a nuclease that degrades single-stranded RNA. It can also be implemented using enzyme preparations derived from bovine pancreas, clearing RNA residues by cleaving phosphodiester bonds between pyrimidine nucleotides.

[0055] In this optional embodiment, the staged treatment strategy achieves a balance between decellularization efficiency and component retention through the sequential regulation of physicochemical actions. The first stage uses an amphoteric detergent to gently remove cell membrane components; its amphiphilic molecular structure binds to the lipid bilayer through hydrophobic interactions, while its hydrophilic groups maintain the hydration of the extracellular matrix. The second stage utilizes the synergistic action of nucleases: deoxyribonuclease I preferentially acts on exposed DNA fragments, while ribonuclease A removes residual RNA molecules. This step-by-step treatment avoids potential interference from simultaneous action of the detergent and nucleases. Isothermal oscillation promotes reagent penetration uniformity through mechanical movement while maintaining the temperature window required for enzyme activity. The gentle treatment with the amphoteric detergent maintains the integrity of the matrix structure, creating suitable conditions for subsequent enzymatic hydrolysis. The staged treatment strategy achieves a synergistic effect between detergent removal and nucleic acid clearance. Common simultaneous treatment methods in the prior art often result in enzyme activity inhibition due to reagent interactions, while step-by-step treatment effectively avoids this problem.

[0056] In some specific embodiments, the amphoteric detergent treatment stage can involve treating with a 0.5% dodecyl dimethyl betaine solution at 37°C with shaking for 6 hours, followed by treatment with a mixed solution containing 50 U / mL deoxyribonuclease I and 20 U / mL ribonuclease A for another 4 hours. In other embodiments, the concentration of the amphoteric detergent can be adjusted to 0.3% to 0.8%, and the nuclease treatment time can be controlled within the range of 3 to 5 hours.

[0057] Another embodiment of the present invention provides the application of a decellularized matrix hydrogel loaded with decellularized animal membrane particles as described above in the field of injectable functional composite biomaterials or implantable functional composite biomaterials.

[0058] Specifically, the injectable application involves drawing 1 mL of the hydrogel precursor solution stored at 4°C into a syringe and injecting it through an 18G needle into the soft tissue defect site (such as subcutaneous ulcers or tendon injuries). The injection volume is adjusted according to the defect volume (usually 0.5 to 2 mL). Avoid strenuous activity for 30 minutes after injection. Apply 0.1 to 0.3T external magnetic field stimulation for 15 minutes daily for one week post-procedure to promote cell recruitment through the mechanical signal transduction of magnetic particles.

[0059] Implantable applications involve cutting pre-gelled hydrogel into a shape that matches the bone defect or cartilage injury site, surgically implanting it into the defect area, and fixing it in place. For the first two weeks post-surgery, magnetic field stimulation is applied three times a week (20 minutes each time), and twice a week from the third to fourth week, to promote mineralization matrix formation and tissue integration. The repair effect is regularly assessed through imaging examinations after implantation, with follow-up typically conducted at 4, 8, and 12 weeks post-surgery.

[0060] The present invention will be further described below with reference to specific embodiments.

[0061] Example 1, Optimization Group.

[0062] Raw material composition: CHAPS (ampholy detergent) 3.0g, DNase I (deoxyribonuclease I) 0.5g, RNase A (ribonuclease A) 0.3g, porcine omentum dECM (decellularized porcine omentum product) 50.0g, decellularized omentum granules 20.0g, hyaluronic acid 2.0g, nanocellulose 3.0g, trypsin 1.0g, collagenase 1.0g, matrix lysin 0.5g, genipin (gardenia extract) 0.3g, chitosan 5.0g, PDA@Fe3O4 (PDA@Fe3O4 magnetic nanoparticles) 0.2g, PGS-P (phosphorylated polyglycerol sebacic acid ester) 2.0g, liposome-encapsulated silver nanoparticles 0.2g, PBS (solvent, phosphate buffer, pH=7.4) to 100g.

[0063] A method for preparing a decellularized matrix hydrogel loaded with decellularized animal reticulum particles includes the following steps: 1. Select 70g of porcine reticulum dECM, cut it into small pieces, add a mixture of amphoteric detergent CHAPS and PBS solvent, and treat it with constant temperature shaking at 37℃. Then add a mixed solution of deoxyribonuclease I and ribonuclease A, and continue shaking treatment at 37℃. Wash with PBS solvent until the amount of residual DNA detected by ultraviolet spectrophotometry is less than the preset threshold to obtain decellularized matrix. 2. Take 20g of the decellularized matrix obtained in step 1 and immerse it in liquid nitrogen. After taking it out, freeze-mill it and sieve it to obtain decellularized membrane particles. Add hyaluronic acid solution and use ultrasonic treatment to modify the surface of the decellularized membrane particles. Add nanocellulose suspension and use 300W ultrasonic treatment to form a functional particle suspension. 3. Take 50g of the decellularized matrix obtained in step 1, add trypsin, collagenase and matrix lysin in a mass ratio and mix them. Add PBS solvent to adjust the solid-liquid ratio to 1:20. Incubate at 37°C in a constant temperature water bath for 35 minutes. Monitor the enzymatic hydrolysis in real time using a dynamic rheometer. When the storage modulus reaches 1.5kPa, add the functional particle suspension obtained in step 2, chitosan solution and gardenia extract solution in sequence. After magnetic stirring, add PDA@Fe3O4 magnetic nanoparticles and phosphorylated polyglycerol sebacic acid ester. After vacuum degassing, obtain the hydrogel precursor solution. 4. Transfer the hydrogel precursor solution obtained in step 3 to the mold, and let it stand in a constant temperature incubator at 37℃ for 8 minutes to complete the initial gelation. Then place it in a magnetic field for 20 minutes to promote the orderly arrangement of magnetic particles and form a decellularized matrix hydrogel with magnetic response characteristics loaded with decellularized animal membrane particles.

[0064] Example 2, High-mechanics group.

[0065] Raw material composition: CHAPS 4.0g, DNase I 0.6g, RNase A 0.4g, porcine reticulum dECM 55.0g, decellularized reticulum granules 25.0g, hyaluronic acid 3.0g, nanocellulose 4.0g, trypsin 1.2g, collagenase 1.2g, matrix lysin 0.6g, genipin 0.4g, chitosan 6.0g, PDA@Fe3O4 0.3g, PGS-P 3.0g, liposome-encapsulated silver nanoparticles 0.3g, PBS to 100g.

[0066] A method for preparing a decellularized matrix hydrogel loaded with decellularized animal reticulum particles includes the following steps: 1. Select 80g of porcine retinoid dECM, cut it into small pieces, add a mixture of amphoteric detergent CHAPS and PBS solvent, and treat with constant temperature shaking at 37℃. Then add a mixed solution of deoxyribonuclease I and ribonuclease A, and continue shaking treatment at 37℃. The total decellularization treatment time is 10 hours. Then rinse with PBS solvent until the residual DNA content detected by ultraviolet spectrophotometry is less than the preset threshold to obtain decellularized matrix. 2. Take 25g of the decellularized matrix obtained in step 1 and immerse it in liquid nitrogen. After taking it out, freeze-mill it and sieve it to obtain decellularized membrane particles. Add hyaluronic acid solution and use ultrasonic treatment to modify the surface of the decellularized membrane particles. Add nanocellulose suspension and use ultrasonic treatment at 300W for 15 minutes to form a functional particle suspension. 3. Take 55g of the decellularized matrix obtained in step 1, add trypsin, collagenase and matrix lysin in a mass ratio and mix them. Add PBS solvent to adjust the solid-liquid ratio to 1:20. Incubate at 37°C in a constant temperature water bath for 45 minutes. Monitor the enzymatic hydrolysis in real time using a dynamic rheometer. When the storage modulus reaches 1.5kPa, add the functional particle suspension obtained in step 2, chitosan solution and gardenia extract solution in sequence. After magnetic stirring, add PDA@Fe3O4 magnetic nanoparticles and phosphorylated polyglycerol sebacic acid ester. After vacuum degassing, obtain the hydrogel precursor solution. 4. Transfer the hydrogel precursor solution obtained in step 3 to the mold, and let it stand in a constant temperature incubator at 37℃ for 10 minutes to complete the initial gelation. Then place it in a magnetic field for 20 minutes to promote the orderly arrangement of magnetic particles and form a decellularized matrix hydrogel with magnetic response characteristics loaded with decellularized animal membrane particles.

[0067] Example 3, High Vitality Group.

[0068] Raw material composition: CHAPS 2.0g, DNase I 0.3g, RNase A 0.2g, porcine reticulum dECM 45.0g, decellularized reticulum granules 15.0g, hyaluronic acid 1.0g, nanocellulose 2.0g, trypsin 0.8g, collagenase 0.8g, matrix lysin 0.3g, genipin 0.2g, chitosan 4.0g, PDA@Fe3O4 0.1g, PGS-P 1.0g, liposome-encapsulated silver nanoparticles 0.1g, PBS to 100g.

[0069] A method for preparing a decellularized matrix hydrogel loaded with decellularized animal reticulum particles includes the following steps: 1. Select 60g of porcine dECM membrane, cut it into small pieces, add a mixture of amphoteric detergent CHAPS and PBS solvent, and treat it with constant temperature shaking at 37℃. Then add a mixed solution of deoxyribonuclease I and ribonuclease A, and continue shaking treatment at 37℃. Wash with PBS solvent until the amount of residual DNA detected by ultraviolet spectrophotometry is less than the preset threshold to obtain decellularized matrix. 2. Take 15g of the decellularized matrix obtained in step 1 and immerse it in liquid nitrogen. After taking it out, freeze-mill it and sieve it to obtain decellularized membrane particles. Add hyaluronic acid solution and use 300W ultrasonic treatment to achieve surface modification of the decellularized membrane particles. Add nanocellulose suspension and use ultrasonic treatment for 15 minutes to form a functional particle suspension. 3. Take 45g of the decellularized matrix obtained in step 1, add trypsin, collagenase and matrix lysin in a mass ratio and mix them. Add PBS solvent to adjust the solid-liquid ratio to 1:20. Incubate at 37°C in a constant temperature water bath for 30 minutes. Monitor the enzymatic hydrolysis in real time using a dynamic rheometer. When the storage modulus reaches 1.5kPa, add the functional particle suspension obtained in step 2, chitosan solution and gardenia extract solution in sequence. After magnetic stirring, add PDA@Fe3O4 magnetic nanoparticles and phosphorylated polyglycerol sebacic acid ester. After vacuum degassing, obtain the hydrogel precursor solution. 4. Transfer the hydrogel precursor solution obtained in step 3 to the mold, let it stand in a constant temperature incubator at 37℃ for 5 minutes to complete the initial gelation, and then place it in a magnetic field for 30 minutes to promote the orderly arrangement of magnetic particles and form a decellularized matrix hydrogel with magnetic response characteristics loaded with decellularized animal membrane particles.

[0070] Comparative Example 1, conventional decellularized group.

[0071] Raw material composition: SDS 2.0g (replacing CHAPS in Example 1), DNase I 0.5g, RNase A 0.3g, porcine reticulum dECM 50.0g, decellularized reticulum granules 20.0g, hyaluronic acid 2.0g, nanocellulose 3.0g, trypsin 1.0g, collagenase 1.0g, matrix lysin 0.5g, genipin 0.3g, chitosan 5.0g, PBS to 100g, and free of magnetic and mineralizing additives.

[0072] The difference between the preparation process and Example 1 is that the decellularization step in step 1 is performed by treating with 1% SDS solution for 12 hours, and the remaining steps are the same as in Example 1, except for the magnetic field treatment process in step 4.

[0073] Comparative Example 1, group without functional additives.

[0074] Raw material composition: CHAPS 3.0g, DNase I 0.5g, RNase A 0.3g, porcine omentum dECM 50.0g, decellularized omentum granules 20.0g, trypsin 1.0g, collagenase 1.0g, matrix lysin 0.5g, glutaraldehyde 0.1g (replacing genipin), PBS to 100g (no nanocellulose, magnetic and mineralizing additives).

[0075] The difference between the preparation process and Example 1 is that the decellularization step in step 1 uses 0.1% glutaraldehyde crosslinking and reacts at 37°C for 2 hours, without the ultrasonic dispersion in step 2 and the magnetic field treatment in step 4.

[0076] Effect Example The decellularized matrix hydrogel products loaded with decellularized animal reticulum particles prepared in Examples 1 to 3 and Comparative Examples 1 to 2 were subjected to mechanical property tests, degradation performance evaluation, cell compatibility experiments, antibacterial performance tests, and bone regeneration effect verification, respectively. The test methods were designed as follows: 1. Mechanical property testing: In accordance with ASTM F2512 standard, the Instron universal testing machine was used to conduct compression tests on each group of hydrogels. The sample size was Φ8mm×5mm, the compression rate was 1mm / min, and 5 samples were tested for each group. The compression modulus and maximum compressive strength were calculated.

[0077] 2. Degradation performance evaluation: The hydrogel samples (Φ10mm×2mm) were immersed in PBS buffer containing 1mg / mL collagenase at 37℃. The weight loss rate was calculated by weighing the samples periodically. Three samples were used in each group, and the degradation data were recorded at 7, 14 and 21 days.

[0078] 3. Cell compatibility assay: L929 fibroblasts were analyzed using the MTT assay. 5 × 10⁶ cells were used. 4 Cells were seeded on the surface of a hydrogel and cultured for 1, 3, and 5 days. The OD value was measured to calculate the relative cell viability. Cell survival status was observed by Live / Dead staining.

[0079] 4. Antibacterial performance test: The plate count method was used, and the hydrogel sample was placed on a 1×10⁻⁶ plate. 6 CFU / mL Escherichia coli and Staphylococcus aureus were co-cultured for 24 hours, and the antibacterial rate was calculated. Each group was repeated 3 times.

[0080] 5. Verification of bone regeneration effect: A rat skull defect model was established. After implantation of hydrogel, samples were taken at 4 and 8 weeks. Bone volume fraction (BV / TV) was calculated by Micro-CT scanning. Six rats were in each group for histological analysis.

[0081] The test results are shown in Table 1.

[0082] Table 1: Performance indicators of Examples 1-3 and Comparative Examples 1-2.

[0083]

[0084] As shown in Table 1, Example 1 exhibited the best overall performance, with a compressive modulus of 920 kPa, significantly higher than Comparative Example 1 (650 kPa) and Comparative Example 2 (780 kPa), indicating the synergistic enhancing effect of the genipin-chitosan crosslinking system and the nanocellulose network. The degradation rate was 45% after 21 days, achieving a time-matched balance between mechanical support and tissue regeneration. In contrast, Comparative Example 2 degraded too slowly (28%) due to glutaraldehyde crosslinking, while Comparative Example 1 degraded too quickly (62%) due to the lack of functional additives.

[0085] In the cell viability test, Example 1 showed a cell viability of 96.2%, significantly higher than Comparative Example 2 (72.8%) which used glutaraldehyde, confirming the biocompatibility advantage of the natural cross-linking agent. Regarding antibacterial properties, the example groups containing liposome silver nanoparticles all exhibited antibacterial rates >95%, significantly superior to the unadded comparative examples.

[0086] Regarding bone regeneration efficacy, the BV / TV value of Example 1 (38.6%) was 71.6% higher than that of Comparative Example 1, validating the effectiveness of the magnetically responsive mineralization system. Comparative Example 2, lacking functional additives, exhibited the worst bone regeneration effect (15.7%). Through comprehensive comparison of various parameters, the hydrogel designed in this scheme demonstrates significant advantages in mechanical properties, degradation controllability, biocompatibility, and tissue regeneration capacity.

[0087] In summary, by employing the above-mentioned technical solutions of this invention, the use of CHAPS amphoteric detergent instead of traditional SDS ensures efficient decellularization while reducing ECM damage. Combined with an automated oscillation system for precise control of processing parameters, it effectively controls fluctuations in collagen content and minimizes DNA residue, significantly improving batch-to-batch consistency. The introduction of a dual dispersion mechanism of hyaluronic acid-modified nanocellulose network, where hyaluronic acid increases particle hydrophilicity through hydroxylation modification and nanocellulose forms a three-dimensional support network through 300W ultrasonic treatment, significantly prolongs particle suspension stability and effectively controls deviations. Furthermore, the synergistic crosslinking system of genipin (gardenia extract) and chitosan achieves precise control over mechanical properties and degradation rate. The biepoxy structure of genipin and the amino group of chitosan form a stable heterocyclic structure, while adjusting the degree of deacetylation of chitosan achieves a controllable degradation cycle of 4-8 weeks, matching the regeneration needs of different tissues. By integrating a magnetic response mineralization system, PDA@Fe3O4 magnetic particles generate mechanical signals under the action of an external magnetic field, which promotes calcium ion influx by activating Piezo1 channels. Combined with the autonomous mineralization ability of PGS-P, it effectively enhances the activity of the bone differentiation marker ALP. The use of liposome encapsulation technology achieves sustained release control of silver nanoparticles, effectively maintaining the antibacterial rate while reducing the cytotoxicity level to below grade 1, effectively solving the contradiction between antibacterial and biocompatibility.

[0088] While the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and all such changes and modifications will fall within the scope of protection of the present invention.

Claims

1. A decellularized matrix hydrogel loaded with decellularized animal reticulum particles, characterized in that, Includes decellularization treatment agents, decellularization matrix, functional particles, enzymatic hydrolysants, cross-linking stabilizers, functional additives, and solvents; The functional additives include PDA@Fe3O4 magnetic nanoparticles, phosphorylated polyglycerol sebacic acid ester, and liposome-encapsulated silver nanoparticles.

2. The decellularized matrix hydrogel loaded with decellularized animal reticulum particles according to claim 1, characterized in that, The decellularization treatment agent includes an amphoteric detergent, deoxyribonuclease I, and ribonuclease A.

3. The decellularized matrix hydrogel loaded with decellularized animal reticulum particles according to claim 1, characterized in that, The decellularized matrix is ​​a decellularized product of porcine omentum or bovine omentum.

4. The decellularized matrix hydrogel loaded with decellularized animal reticulum particles according to claim 1, characterized in that, The functional particles include decellularized membrane particles, hyaluronic acid, and nanocellulose, wherein the decellularized membrane particles are derivative products obtained by freeze-drying and grinding a portion of the decellularized matrix.

5. The decellularized matrix hydrogel loaded with decellularized animal reticulum particles according to claim 4, characterized in that, The decellularized membrane particles have a diameter of less than 50 μm; the hyaluronic acid has a molecular weight of 700 kDa to 900 kDa; and the nanocellulose is eucalyptus pulp (2,2,6,6-tetramethylpiperidine-1-oxygen radical) oxidized nanocellulose or bacterial nanocellulose.

6. The decellularized matrix hydrogel loaded with decellularized animal reticulum particles according to claim 1, characterized in that, The enzymatic hydrolysants include pancreatin, collagenase, and matrix lysin.

7. The decellularized matrix hydrogel loaded with decellularized animal reticulum particles according to claim 1, characterized in that, The cross-linking stabilizer includes gardenia extract and chitosan.

8. A method for preparing a decellularized matrix hydrogel loaded with decellularized animal reticulum particles as described in any one of claims 1-7, characterized in that, Includes the following steps: S1: Select animal reticulum tissue, cut it into small pieces, add decellularization agent and solvent, and treat it at a constant temperature of 36 to 38°C with shaking. Then rinse it with solvent buffer to obtain decellularized matrix. S2: Take a portion of the decellularized matrix obtained in step S1 and immerse it in liquid nitrogen. After taking it out, perform freeze grinding and sieve to obtain decellularized membrane particles. Add hyaluronic acid solution and use ultrasonic treatment to modify the surface of the decellularized membrane particles. Add nanocellulose suspension and use ultrasonic treatment to form a functional particle suspension. S3: Take the decellularized matrix obtained in the remaining step S1, add an enzymatic hydrolysant and mix, add a solvent to adjust the solid-liquid ratio to 1:15 to 25, and enzymatically hydrolyze in a constant temperature water bath at 36 to 38°C. Monitor in real time with a dynamic rheometer. When the storage modulus is not lower than 1.5 kPa, terminate the enzymatic hydrolysis. Add the functional particle suspension obtained in step S2 and the crosslinking stabilizer in sequence. After magnetic stirring, add the functional additives. After vacuum degassing, obtain the hydrogel precursor solution. S4: Transfer the hydrogel precursor solution obtained in step S3 into a mold, and let it stand in a constant temperature incubator at 36 to 38°C to complete the initial gelation. Then place it in a magnetic field to promote the orderly arrangement of magnetic particles and form a decellularized matrix hydrogel with magnetic response characteristics loaded with decellularized animal membrane particles.

9. The method for preparing a decellularized matrix hydrogel loaded with decellularized animal membrane particles according to claim 8, characterized in that, The decellularization treatment agent includes an amphoteric detergent, deoxyribonuclease I, and ribonuclease A. Step S1, which involves adding the decellularization treatment agent and solvent and treating with constant temperature shaking at 36 to 38°C, includes: adding a mixture of the amphoteric detergent and the solvent and treating with constant temperature shaking at 36 to 38°C; then adding a mixed solution of deoxyribonuclease I and ribonuclease A and continuing to treat with shaking at 36 to 38°C.

10. The application of a decellularized matrix hydrogel loaded with decellularized animal membrane particles as described in any one of claims 1-7 in the field of injectable functional composite biomaterials or implantable functional composite biomaterials.

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