A tissue regeneration filler, and a preparation method and application thereof

Abstract

The application discloses a kind of tissue regeneration fillers and preparation method and application thereof, belong to the field of biomedical materials and medical cosmetology technical field. Including the following mass fraction components: mixed microparticle 1.0~5.0 parts;Mixed microparticle includes acellular matrix microparticle and basement membrane microparticle, and the mass ratio of acellular matrix microparticle and basement membrane microparticle is 10‑1:1.The application extracts and compounding two kinds of microparticles respectively, combines gel matrix and dispersion liquid processing, retains the scaffold function of acellular matrix, supplements basement membrane specific active ingredient, realizes instant support and long-acting regeneration synergy;Filler has excellent injectability and biocompatibility, can accurately repair basement membrane structure degradation, promote cell directional migration and tissue functional regeneration, and is widely used in facial rejuvenation, wound repair and soft tissue regeneration.

Description

Technical Field

[0001] This invention relates to the fields of biomedical materials and medical aesthetics, and particularly to a tissue regeneration filler, its preparation method, and its application. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] With the development of medical aesthetics and regenerative medicine, injectable fillers are widely used in the field of facial rejuvenation to achieve tissue repair and volume restoration. Currently, mainstream filler technologies are mainly divided into three categories: physical filler materials, such as hyaluronic acid and collagen, achieve immediate filling through space-occupying effects, but cannot reverse the intrinsic aging process of tissues and require repeated injections; collagen-stimulating materials, such as poly-L-lactic acid, induce fibrosis through foreign body reaction, but the newly formed tissue structure is disordered, easily leading to abnormal texture and nodules; and biological scaffold materials, such as decellularized matrix, although providing a three-dimensional scaffold to support cell ingrowth, have a single composition, mainly type I / III collagen, and cannot specifically repair the core aspects of skin aging, such as basement membrane degeneration. The basement membrane, as a nanoscale precision structure in the dermal-epidermal junction, is assembled from molecules such as type IV collagen and laminin according to a specific topological conformation. It not only maintains epidermal cell polarity but also regulates fibroblast metabolism through integrin signaling. Skin aging is accompanied by the loss of basement membrane components and structural disintegration, leading to epidermal atrophy, weakened dermal-epidermal adhesion, and abnormal mechanical signal transduction.

[0004] While existing decellularized matrix materials have made progress in macroscopic structural simulation, their preparation process generally leads to the loss or inactivation of characteristic components of the basement membrane. This results in the materials only functioning as ordinary scaffolds after implantation, failing to reconstruct the biological communication mechanism at the dermal-epidermal interface. Therefore, how to preserve the activity of basement membrane-specific components and effectively integrate them into the filling system has become a key theoretical problem in overcoming the bottlenecks of existing regenerative technologies. Summary of the Invention

[0005] In view of this, the present invention provides a tissue regeneration filler, its preparation method, and its application, which maintains the uniform suspension of microparticles while ensuring good injectability and biocompatibility, and coordinates the timely release of support and regeneration signals. The repair effect provided by the filler can precisely target the functional decline of the basement membrane, a key structure, and simultaneously create a suitable cellular microenvironment at the implantation site, enabling host cells to complete targeted differentiation. The technical solution provided by the present invention is as follows: In a first aspect, the present invention provides a tissue regeneration filler comprising the following components in parts by weight: 1.0 to 5.0 parts of mixed microparticles; The mixed microparticles comprise decellularized matrix microparticles and basement membrane microparticles, wherein the mass ratio of decellularized matrix microparticles to basement membrane microparticles is 10⁻¹:1.

[0006] Decellularized matrix microparticles and basement membrane microparticles work together in fillers to synergistically promote cell adhesion, directed migration, and tissue-specific regeneration. Decellularized matrix microparticles, acting as a natural three-dimensional scaffold, provide macroscopic mechanical support and space for cell ingrowth, ensuring immediate volume maintenance and providing a foundation for tissue remodeling. Basement membrane microparticles, through a separate extraction process, retain specialized active ingredients such as type IV collagen, laminin, and nestin. Laminin can bind to integrin receptors on the surface of epidermal basal cells and dermal fibroblasts, activating signaling pathways to regulate directed cell differentiation. Type IV collagen can rebuild the unique reticular structure of the basement membrane, repairing the adhesion stability of the dermal-epidermal junction. Nestin can assist vascular endothelial cell migration, providing a nutrient pathway for regenerated tissue. The two are combined in a ratio of 10:1 to 1:1, which avoids the problems of insufficient signaling molecules and low regeneration efficiency caused by excessive decellularized matrix, and also prevents the defects of insufficient mechanical strength of the scaffold and immediate support failure caused by excessive basement membrane microparticles. Ultimately, it achieves synergy between scaffold support and signal transduction, upgrading the filler from simple morphological filling to functional regeneration of epidermal-dermal synergistic repair, effectively improving aging-related problems such as epidermal atrophy and abnormal mechanical signal transduction.

[0007] Preferably, the particle size range of the decellularized matrix microparticles is 10 μm to 300 μm; and the particle size range of the basement membrane microparticles is 1 to 100 μm.

[0008] In facial filler applications, microparticles of different sizes work synergistically to achieve perfect results: ≤25μm microparticles primarily promote rapid biorecognition and angiogenesis, providing a nutritional basis for tissue regeneration; 25-50 μm microparticles focus on inducing fibroblast migration and autologous collagen regeneration, achieving long-lasting tissue reconstruction; 50-100 μm microparticles act as the mechanical main body, providing immediate and lasting physical support to maintain volumetric filling effects; and 100-300 μm microparticles guide spatial occupancy and orderly tissue remodeling in deep tissues, preventing deformation. Through individual or combined use, the multi-level particle size system achieves complementary degradation timing and functions, thus simultaneously completing the integrated synergistic enhancement of short-term physical filling and long-term tissue regeneration.

[0009] Preferably, the mass ratio of the decellularized matrix microparticles to the basement membrane microparticles is 5-3:1.

[0010] Decellularized matrix (dECM) refers to the fibrous network structure of extracellular matrix (exosomes) (ECM) with relatively weak immunogenicity remaining after human or animal tissues from different sources have undergone decellularization processes to remove highly antigenic components from the tissue. Further, the decellularized matrix microparticles are selected from one or more of the following: skin, small intestine, diaphragm, meninges, pericardium, fascia, nerves, kidneys, placenta, bladder, blood vessels, greater omentum, tendons, and muscles, preferably the small intestine.

[0011] Preferably, the tissue regeneration filler further comprises the following components in parts by weight: 1.0 to 8.0 parts of dispersion, 0.5 to 8.0 parts of osmotic pressure regulator, 60.0 to 95.0 parts of pH buffer, and 1.0 to 30.0 parts of gel forming agent; The dispersion is selected from at least one of physiological saline, glycerol, propylene glycol, polyethylene glycol, Tween 80, and polysorbate 60; The osmotic pressure regulator is selected from one or more of trehalose, mannitol, sorbitol, sodium chloride, and sucrose; The pH buffer solution is selected from at least one of the following: acetate-sodium acetate buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer, citrate-sodium citrate buffer, and hydroxyethylpiperazine ethanesulfonic acid buffer. The gelling agent is selected from one or more of sodium hyaluronate, cross-linked sodium hyaluronate, methylcellulose, collagen, povidone, chitosan, agarose, and sodium alginate.

[0012] As a continuous phase, the gel-forming agent not only serves as a stable carrier for the dispersed phase microparticles but also possesses a certain spatial occupancy capacity, enabling rapid volume filling after implantation. Simultaneously, its slow degradation characteristics and the regenerative effect of the mixed microparticles create a time-series match: in the early stages, the gel provides immediate morphological maintenance; in the mid-to-late stages, decellularized matrix microparticles guide host cell ingrowth and construct a new tissue scaffold; and basement membrane microparticles continuously transmit regeneration signals, promoting the differentiation of new tissue into physiological structures. This synergistic mode of immediate gel support and long-term microparticle regeneration avoids the drawbacks of repeated injections required by physical fillers and overcomes the morphological instability during the regeneration cycle of traditional scaffold materials, achieving an integration of short-term morphological improvement and long-term tissue repair.

[0013] This invention utilizes a biocompatible gel matrix as the continuous phase, along with a gel-forming agent, an osmotic pressure regulator, and a pH buffer to ensure a high degree of compatibility between the filler's physicochemical properties and human tissue fluid. The biocompatible gel matrix reduces the risk of immune rejection in vivo and enhances tissue adaptability; the osmotic pressure regulator and pH buffer maintain system stability, preventing local tissue irritation due to osmotic imbalance or pH abnormalities, further ensuring the safety of clinical applications. Simultaneously, this system is compatible with the addition of anesthetics and nutrients, meeting clinical analgesia needs while providing nutritional support for tissue regeneration, thus broadening the product's clinical application scenarios.

[0014] Preferably, the tissue regeneration filler further comprises one or more of an anesthetic and a nutrient, wherein the anesthetic is present in a mass fraction of 0.05 to 1 part and the nutrient is present in a mass fraction of 0.01 to 1 part; The anesthetic agent is selected from any one of lidocaine hydrochloride, prilocaine, procaine, benzocaine, bupivacaine, and tetracaine; The nutrients are selected from one or more of the following: amino acids, vitamins, polynucleotides, polynucleotides, coenzymes, minerals, trace elements, nucleic acids, glutathione, and polypeptides.

[0015] Secondly, the present invention also provides a method for preparing the tissue regeneration filler described in the first aspect, comprising the following steps: S1. Basement membrane microparticles were prepared by sequentially pretreating biological tissues, mechanically separating them, decellularizing them, freeze-drying them, inactivating pathogens, and grinding them at ultra-low temperatures. S2. Mix the basement membrane microparticles obtained in step S1 with the decellularized matrix microparticles and dispersion to form a dispersed phase; S3. Mix the gel forming agent, osmotic pressure regulator and pH buffer solution, stir well, and prepare the gel matrix; Preferably, in step S3, after stirring evenly, one or more of the anesthetic and nutrient agents are added and mixed evenly. S4. The dispersed phase obtained in step S2 is mixed with the gel matrix to obtain a tissue regeneration filler.

[0016] This invention effectively solves the technical problems of basement membrane structure damage and loss of functional components during the extraction and preparation of traditional decellularized matrix materials by preparing basement membrane microparticles separately and then compounding them with decellularized matrix microparticles. This compounding strategy not only preserves the scaffold function of the decellularized matrix but also reconstructs a bioactive matrix-basement membrane composite microenvironment by supplementing complete basement membrane components.

[0017] Adding dry microparticles directly to a viscous gel matrix can easily lead to agglomeration due to their high surface energy, hydrophobicity, or intermolecular forces, forming difficult-to-break aggregates. This not only causes particle sedimentation, affecting product uniformity, but can also clog needles, impacting the injection experience. Pre-dispersing and pre-treating the mixed microparticles with a dispersion solution effectively prevents agglomeration during subsequent mixing with the gel. The dispersion solution reduces the surface energy of the microparticles and weakens intermolecular forces through pre-treatment, ensuring uniform suspension of decellularized matrix microparticles and basement membrane microparticles in the continuous phase. This avoids poor product uniformity caused by particle sedimentation and prevents agglomerated particles from clogging the injection needle, significantly improving injection smoothness. Furthermore, traditional processes often require high-intensity mechanical forces (such as high-speed grinding or severe shearing) to break up agglomerates when directly mixing microparticles with the gel. This process can easily generate localized high temperatures and high shear stress, leading to denaturation and inactivation of fragile active proteins in the basement membrane. Pre-dispersion treatment, however, can be performed under gentler conditions, avoiding high-intensity mechanical forces.

[0018] Preferably, in step S1, the method for preparing basement membrane microparticles includes the following steps: (1) Mechanical layering: The pretreated biological tissue is sequentially peeled off the serosa and outer muscle layer, and the mucosal surface of the remaining tissue is treated with collagenase and trypsin. After treatment, it is rinsed and then peeled off to obtain the target tissue layer. (2) Gradient decellularization: The target tissue layer was successively soaked and shaken in Tris-HCl solution, then in sodium deoxycholate solution containing PMSF, and then in a mixture of Tris-HCl containing DNase I and RNase A. After each shaking treatment, the tissue was rinsed. (3) Post-processing: The decellularized target tissue layer was freeze-dried, irradiated with gamma rays, and ground and sieved in liquid nitrogen.

[0019] Preferably, in step (1), the pretreatment is as follows: the biological tissue is squeezed and rinsed 3-7 times in sterile physiological saline, transferred to PBS containing 0.1-0.2 mg / mL amoxicillin and 0.05-0.1 mg / mL gentamicin, and stored at 4-6℃ for 12-24h, with the antibiotic solution being replaced every 6h; In step (1), the biological tissue is selected from one or more of the following: bladder, retina, placental amnion, corneal posterior elastic layer, cerebral blood vessels, and renal tubules, preferably the bladder; In step (1), the concentration of collagenase is 0.01%-0.1%, the concentration of trypsin is 0.01%-0.5%, and the enzyme treatment time is 5-15 min.

[0020] Preferably, in step (2), the mixture is shaken at 3-5°C for 12-24 h in a 5-10 mM Tris-HCl solution; shaken at room temperature for 24-36 h in a 0.5-2% sodium deoxycholate solution containing 1 mM MPMSF; shaken at 35-40°C for 2-4 h in a Tris-HCl mixture containing 50 U / mL DNase I and 50 U / mL RNase A; and rinsed with PBS 4-7 times after each shaking treatment, and rinsed with ultrapure water until no foam is present after the last shaking treatment. In step (3), the freeze drying is performed as follows: first, pre-freezing at -60~-80℃ for 4~6h, then primary drying at -40~-60℃ for 12~24h under vacuum conditions <10Pa, and secondary drying at 25~30℃ for 6~12h; the dose of the γ-ray irradiation is 8~15kGy.

[0021] Thirdly, the present invention also provides the application of the tissue regeneration filler described in the first aspect or the tissue regeneration filler prepared by the preparation method described in the second aspect in the preparation of medical aesthetic materials; Preferably, the dosage form of the material is a gel or a lyophilized agent, and more preferably a gel.

[0022] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) In the tissue regeneration filler of the present invention, the decellularized matrix microparticles and basement membrane microparticles are combined to form mixed microparticles, and in synergy with the biocompatible gel forming agent, the three-dimensional scaffold function of the decellularized matrix microparticles is retained, and the basement membrane microparticles extracted separately are supplemented with specialized active ingredients such as type IV collagen and laminin. These ingredients can bind to the receptors on the surface of epidermal basal cells and dermal fibroblasts, and reconstruct a bioactive matrix-basement membrane composite microenvironment, accurately repairing the degenerative changes of the basement membrane, and solving the defect of existing decellularized matrix materials that can only fill the scaffold and cannot achieve functional regeneration.

[0023] (2) The tissue regeneration filler of the present invention has excellent injectability and biocompatibility, which can meet the requirements of smooth clinical injection operation, while ensuring that the material has good tissue adaptability and safety in vivo.

[0024] (3) The basement membrane component introduced in this invention can significantly improve the local tissue microenvironment, promote cell adhesion, proliferation and orderly arrangement, thereby providing a more favorable biological basis for tissue regeneration and repair. The basement membrane component has the ability to promote angiogenesis, which can guide the host tissue to achieve a rapid and orderly angiogenesis process, broadening the application potential of this material in a variety of fields such as anti-aging, scar repair, wound healing and soft tissue regeneration, and showing a wide range of clinical application prospects.

[0025] (4) The preparation method of the present invention extracts decellularized matrix microparticles and basement membrane microparticles separately, and uses gradient decellularization, γ-ray irradiation and other processes to effectively preserve the specific component activity of the basement membrane, ensuring the safety and functionality of the product and providing a reliable basis for tissue regeneration.

[0026] (5) The tissue regeneration filler of the present invention can be formed in two forms: gel and lyophilized powder, providing a flexible choice for different clinical application scenarios, facilitating dosage form adaptation according to specific treatment needs, and enhancing the applicability of the product. Attached Figure Description

[0027] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0028] Figure 1 These are photographs of the tissue regeneration filler prepared according to the present invention, wherein a is Example 1, b is Example 2, c is Example 3, and d is Example 4; Figure 2 This is an RT-PCR detection of the expression of type IV collagen (Col4a1), laminin (Lamb1), and type I collagen (Col1a1) genes after subcutaneous injection of the tissue regeneration filler prepared in Example 2 of this invention and the fillers in Comparative Examples 1-3 in animal experiments. Figure 3 This is an RT-PCR detection of the expression of type IV collagen (Col4a1), laminin (Lamb1), and type I collagen (Col1a1) genes one month after subcutaneous injection of the tissue regeneration filler prepared in Examples 1-5 of this invention in animal experiments. Figure 4 This is an RT-PCR detection of the expression of type IV collagen (Col4a1), laminin (Lamb1), and type I collagen (Col1a1) genes in animal experiments after subcutaneous injection of the tissue regeneration filler prepared in Examples 1-5 of this invention for 6 months. Figure 5 This is an RT-PCR detection of the expression of transforming growth factor (TGF-β1) and vascular endothelial growth factor (VEGF) genes after subcutaneous injection of the tissue regeneration filler prepared in Examples 1-5 of this invention and Comparative Examples 1-3 in animal experiments. Figure 6 These are images of type IV collagen staining 7 days after subcutaneous injection of the tissue regeneration filler of Example 2 of this invention in animal experiments. Detailed Implementation

[0029] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0030] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0031] The PBS, ice-cold PBS, and PBS solution used below have a pH of 7.0.

[0032] The decellularized matrix microparticles used in this invention embodiment are obtained from the submucosa of the small intestine after washing and trimming, acetic acid chopping, decellularization, nucleic acid removal and washing, drying, ultra-low temperature grinding and grading, and pathogen inactivation. The specific preparation method is as follows: (1) Raw material cleaning and trimming: Take fresh pig small intestine, rinse it with ice-cold PBS to remove the contents, cut it lengthwise, and rinse it clean with PBS solution.

[0033] (2) Coarse cutting: Cut the cleaned whole-wall small intestine (including the mucosa, submucosa, muscle layer and serosa) directly into small pieces of about 1 cm × 1 cm.

[0034] (3) Decellularization: The tissue block (the small block after step (2)) was completely immersed in 20 times its volume of 1.0% sodium dodecyl sulfate (SDS) solution and placed on a constant temperature shaker at 37°C for continuous high-speed shaking at 150 rpm for 72 hours. Fresh SDS solution was replaced every 24 hours during this period.

[0035] (4) Nucleic acid removal and washing: Discard the SDS solution, transfer the tissue block to 1.0 M sodium chloride PBS solution, treat on a shaker at room temperature for 6 hours, rinse with plenty of deionized water, and then transfer to flowing deionized water for continuous rinsing for 48 hours, changing the water multiple times until the washing solution is clear, foam-free, and neutral as determined by pH test paper, to ensure that SDS and other reagents are completely removed.

[0036] (5) Freeze-drying: Spread the matrix (the material treated in step (4)) evenly on a freeze-drying tray and pre-freeze at -75℃ for 4.5h. Primary drying: -45℃, vacuum degree 4Pa, for 18h. Secondary drying: 27℃, vacuum degree 2Pa, for 8h.

[0037] (6) Cryogenic grinding and grading: Place the mortar, which has been pre-cooled by liquid nitrogen for 30 min, in a sterile laminar flow hood, add the lyophilized decellularized matrix (the matrix after treatment in step (5)), and continuously replenish with liquid nitrogen. Grind with a sterile pestle until powdery, replenish with liquid nitrogen every 30 s during the grinding process, sieve sequentially, collect the microparticles, and load them into centrifuge tubes.

[0038] (7) Pathogen inactivation: The preparation was completed by irradiation with cobalt-60 gamma rays at a dose of 25.0 kGy.

[0039] In addition to the methods described above, decellularized matrix microparticles can also be prepared using other conventional operations known to those skilled in the art, or commercially available decellularized matrix microparticles can be purchased.

[0040] The cross-linked sodium hyaluronate used in this embodiment of the invention is obtained by adding sodium hyaluronate to a cross-linking agent and an alkaline solution to carry out a cross-linking reaction, then adding an acid solution to neutralize to neutral, and finally purifying and swelling. The specific preparation method is as follows: Weigh 2.0 g of sodium hyaluronate (1500 kDa molecular weight) into a reaction vessel, add 0.8 g of BDDE (crosslinking agent to sodium hyaluronate mass ratio of 0.4:1) and a pH 10.5 NaOH solution (BDDE mass concentration in alkaline solution is 2%, i.e., 0.8 g BDDE dissolved in 40 mL of 0.1 mol / L NaOH solution), mix thoroughly, and stir at 40 °C for 4 hours to form a crosslinked gel. After the reaction is complete, adjust the pH of the reaction solution to neutral with 1 mol / L HCl solution to terminate the crosslinking reaction. Transfer to 50 mmol / L HEPES buffer, stir and swell at 25 °C for 12 hours to allow the gel to fully absorb water and swell to a total mass of 80.0 g, then granulate and sieve to obtain the final product.

[0041] In addition to the methods described above, cross-linked sodium hyaluronate can also be prepared using other conventional operations known to those skilled in the art, or commercially available cross-linked sodium hyaluronate can be purchased.

[0042] I. Example of preparation of basement membrane microparticles Preparation Example 1: Preparation of Basement Membrane Microparticles (1) Raw material pretreatment: Take the bladder of a healthy pig, aseptically separate it within 1 hour after slaughter, and remove the ureteral stump and surrounding adipose tissue. Immerse it in sterile physiological saline at 4°C and squeeze and rinse 5 times to remove urine. Transfer it to PBS containing 0.15 mg / mL amoxicillin and 0.075 mg / mL gentamicin, and store it in a sealed refrigerator at 4°C for 20 hours. Replace it with fresh antibiotic PBS (i.e. the above-mentioned PBS containing amoxicillin and gentamicin) at 6 hours, 12 hours and 18 hours respectively. Before each replacement, quickly rinse the surface of the bladder with sterile PBS once to avoid old solution residue.

[0043] (2) Mechanical separation: In a sterile laminar flow hood, sterile dissecting forceps were used to sequentially peel off the transparent, thin-film-like serosal layer and the light pink fibrous outer muscle layer along the gap between the bladder serosal layer and the outer muscle layer, obtaining the remaining tissue containing the submucosa and mucosa. The remaining tissue was laid flat and fixed on a silicone plate with the mucosa side facing up. Filter paper soaked in 0.1% collagenase and 0.05% trypsin was applied to the mucosa surface of the area to be separated. After 10 minutes of treatment, the filter paper was immediately removed, and the tissue was rinsed repeatedly 5 times with a large amount of sterile PBS at 4°C. At one corner of the tissue slide, the edge of the mucosa was slowly and steadily lifted with micro-forceps. Another flat-tipped forceps was placed close to the bottom of the lifted part and pushed horizontally in a direction parallel to the tissue surface and at an angle of <15°, pushing forward, backward, and to the sides in small amplitudes. Along the gaps between the tissues, the lifting and pushing operations were alternately performed to peel off the submucosa and mucosa, obtaining a basement membrane composite layer containing part of the submucosa.

[0044] (3) Gradient decellularization: The basement membrane composite layer obtained in step (2) was immersed in 7.5 mM Tris-HCl (pH 8.0) and shaken on a constant temperature shaker at 4°C for 18 h. After shaking, it was rinsed 5 times with sterile PBS. Then, it was shaken at room temperature for 30 h in 1.0% sodium deoxycholate (containing 1 mM PMSF). After shaking, it was rinsed 5 times with sterile PBS until the rinsing solution was clear. It was then transferred to sterile 0.1 M Tris-HCl (pH 8.0) containing DNase I (50 U / mL) and RNase A (50 U / mL) and shaken at 37°C for 3 h. After treatment, it was rinsed 5 times with sterile PBS and then rinsed with sterile ultrapure water until the rinsing solution was free of foam.

[0045] (4) Freeze-drying: The matrix (the substance treated in step (3)) is spread in a single layer on a freeze-drying tray, pre-frozen at -70℃ for 5h, primary drying: -50℃, vacuum degree 5Pa, for 20h; secondary drying: 28℃, vacuum degree 3Pa, for 9h; after freeze-drying, a white porous substrate membrane is obtained.

[0046] (5) Pathogen inactivation: The freeze-dried basement membrane was placed in a sterile irradiation bag and irradiated with cobalt-60 γ rays at a dose of 8.0 kGy.

[0047] (6) Cryogenic grinding and grading: Under aseptic conditions, a mortar pre-cooled by liquid nitrogen for 30 min was placed in a sterile laminar flow hood, and an irradiated lyophilized basement membrane was added, with liquid nitrogen continuously replenished. The membrane was ground into powder using a sterile pestle, with liquid nitrogen replenished every 30 s during the grinding process. The membrane was then sieved sequentially to collect the basement membrane particles, which were then placed into sterile centrifuge tubes for later use.

[0048] Preparation Example 2: Preparation of Basement Membrane Microparticles (1) Raw material pretreatment: Take the bladder of healthy pigs, aseptically separate it within 1 hour after slaughter, and remove the ureteral stump and surrounding adipose tissue. Immerse it in sterile physiological saline at 4℃ and squeeze and rinse 5 times to remove urine. Transfer it to PBS containing 0.1 mg / mL amoxicillin and 0.05 mg / mL gentamicin, and store it in a sealed refrigerator at 4℃ for 12 hours. Replace it with fresh antibiotic PBS (i.e. the above-mentioned PBS containing amoxicillin and gentamicin) every 6 hours.

[0049] (2) Mechanical separation: In a sterile laminar flow hood, sterile dissecting forceps were used to sequentially peel off the transparent, thin-film-like serosal layer and the light pink fibrous outer muscle layer along the gap between the bladder serosal layer and the outer muscle layer, obtaining the remaining tissue containing the submucosa and mucosa. The remaining tissue was laid flat and fixed on a silicone plate with the mucosa side facing up. Filter paper soaked in 0.05% collagenase and 0.25% trypsin was applied to the mucosa surface of the area to be separated. After 5 min of treatment, the filter paper was immediately removed, and the tissue was rinsed repeatedly 5 times with a large amount of sterile PBS at 4°C. At one corner of the tissue slide, the edge of the mucosa was slowly and steadily lifted with micro-forceps. Another flat-tipped forceps was placed close to the bottom of the lifted part and pushed horizontally in a direction parallel to the tissue surface and at an angle of <15°, pushing forward, backward, and to the sides in small amplitudes. Along the gaps between the tissues, the lifting and pushing operations were alternately performed to peel off the submucosa and mucosa, obtaining a basement membrane composite layer containing part of the submucosa.

[0050] (3) Gradient decellularization: The basement membrane composite layer obtained in step (2) was immersed in 5 mM Tris-HCl (pH 8.0) and shaken on a constant temperature shaker at 4°C for 12 h. After shaking, it was rinsed 5 times with sterile PBS. Then, it was shaken at room temperature for 24 h in 0.5% sodium deoxycholate (containing 1 mM MPMSF). After shaking, it was rinsed 5 times with sterile PBS until the rinsing solution was clear. It was then transferred to sterile 0.1 M Tris-HCl (pH 8.0) containing DNase I (50 U / mL) and RNase A (50 U / mL) and shaken at 37°C for 2 h. After treatment, it was rinsed 5 times with sterile PBS and then rinsed with sterile ultrapure water until the rinsing solution was free of foam.

[0051] (4) Freeze-drying: The matrix (the substance treated in step (3)) is spread in a single layer on a freeze-drying tray and pre-frozen at -60℃ for 6 hours. Primary drying: -40℃, vacuum degree 8Pa, for 12 hours; secondary drying: 25℃, vacuum degree 6Pa, for 6 hours.

[0052] (5) Pathogen inactivation: Cobalt-60 γ-ray irradiation was used with an irradiation dose of 15.0 kGy.

[0053] (6) Cryogenic grinding and grading: Under aseptic conditions, a mortar pre-cooled by liquid nitrogen for 30 min was placed in a sterile laminar flow hood, and an irradiated lyophilized basement membrane was added, with liquid nitrogen continuously replenished. The membrane was ground into powder using a sterile pestle, with liquid nitrogen replenished every 30 s during the grinding process. The membrane was then sieved sequentially to collect the basement membrane particles, which were then placed into sterile centrifuge tubes for later use.

[0054] Preparation Example 3: Preparation of Basement Membrane Microparticles (1) Raw material pretreatment: Take the bladder of a healthy pig, aseptically separate it within 1 hour after slaughter, and remove the ureteral stump and surrounding adipose tissue. Immerse it in sterile physiological saline at 4℃ and squeeze and rinse 5 times to remove urine. Transfer it to PBS containing 0.1 mg / mL amoxicillin and 0.05 mg / mL gentamicin, and store it in a sealed refrigerator at 4℃ for 12 hours. Replace it with fresh antibiotic PBS (i.e. the above-mentioned PBS containing amoxicillin and gentamicin) every 6 hours. Before replacement, use sterile PBS to quickly rinse the surface of the bladder once to avoid old solution residue.

[0055] (2) Mechanical separation: In a sterile laminar flow hood, sterile dissecting forceps were used to sequentially peel off the transparent, thin-film-like serosal layer and the light pink fibrous outer muscle layer along the gap between the bladder serosal layer and the outer muscle layer, obtaining the remaining tissue containing the submucosa and the mucosa. The remaining tissue was laid flat and fixed on a silicone plate with the mucosa side facing up. Filter paper soaked in 0.01% collagenase and 0.01% trypsin was placed on the mucosa surface of the area to be separated. After 15 minutes of treatment, the filter paper was immediately removed, and the tissue was rinsed repeatedly 5 times with a large amount of sterile PBS at 4°C. At one corner of the tissue slide, the edge of the mucosa was slowly and steadily lifted with micro-forceps. Another flat-tipped forceps was placed close to the bottom of the lifted part and pushed horizontally in a direction parallel to the tissue surface and at an angle of <15°, pushing forward, backward, and to the sides in small amplitudes. Along the gaps between the tissues, the lifting and pushing operations were alternately performed to peel off the submucosa and the mucosa, obtaining a basement membrane composite layer containing part of the submucosa.

[0056] (3) Gradient decellularization: The basement membrane composite layer obtained in step (2) was immersed in 10 mM Tris-HCl (pH 8.0) and shaken on a constant temperature shaker at 4°C for 24 h. After shaking, it was rinsed 5 times with sterile PBS. Then, it was shaken at room temperature for 36 h with 2% sodium deoxycholate (containing 1 mM PMSF). After shaking, it was rinsed 5 times with sterile PBS until the rinsing solution was clear. It was then transferred to sterile 0.1 M Tris-HCl (pH 8.0) containing DNase I (50 U / mL) and RNase A (50 U / mL) and shaken at 37°C for 4 h. After treatment, it was rinsed 5 times with sterile PBS and then rinsed with sterile ultrapure water until the rinsing solution was free of foam.

[0057] (4) Freeze-drying: The matrix (the substance treated in step (3)) is spread in a single layer on a freeze-drying tray and pre-frozen at -80℃ for 4 hours. Primary drying: -60℃, vacuum degree 2Pa, for 24 hours; secondary drying: 30℃, vacuum degree 1Pa, for 12 hours.

[0058] (5) Pathogen inactivation: Cobalt-60 γ-ray irradiation was used with an irradiation dose of 12.0 kGy.

[0059] (6) Cryogenic grinding and grading: Under aseptic conditions, a mortar pre-cooled by liquid nitrogen for 30 min was placed in a sterile laminar flow hood, and an irradiated lyophilized basement membrane was added, with liquid nitrogen continuously replenished. The membrane was ground into powder using a sterile pestle, with liquid nitrogen replenished every 30 s during the grinding process. The membrane was then sieved sequentially to collect the basement membrane particles, which were then placed into sterile centrifuge tubes for later use.

[0060] Preparation Example 4: Preparation of Basement Membrane Microparticles (1) Raw material pretreatment: Take the bladder of healthy pigs, aseptically separate it within 1 hour after slaughter, and remove the ureteral stump and surrounding adipose tissue. Immerse it in sterile physiological saline at 4℃ and squeeze and rinse 5 times to remove urine. Transfer it to PBS containing 0.18 mg / mL amoxicillin and 0.09 mg / mL gentamicin, and store it in a sealed refrigerator at 4℃ for 18 hours. Replace it with fresh antibiotic PBS (i.e. the above-mentioned PBS containing amoxicillin and gentamicin) at 6 hours and 12 hours respectively. Before each replacement, use sterile PBS to quickly rinse the surface of the pig small intestine once to avoid old solution residue.

[0061] (2) Mechanical separation: In a sterile laminar flow hood, sterile dissecting forceps are used to sequentially peel off the transparent, thin-film-like serosal layer and the light pink fibrous outer muscle layer along the gap between the bladder serosal layer and the outer muscle layer, obtaining the remaining tissue containing the submucosa and mucosa. The remaining tissue is laid flat and fixed on a silicone plate with the mucosa side facing up. Filter paper soaked in 0.1% collagenase and 0.5% trypsin is applied to the mucosa surface of the area to be separated. After 5 minutes of treatment, the filter paper is immediately removed, and the tissue is rinsed repeatedly 5 times with a large amount of sterile PBS at 4°C. At one corner of the tissue slide, the edge of the mucosa is slowly and steadily lifted with micro-forceps. Another flat-tipped forceps is placed close to the lower part of the lifted portion and pushed horizontally in a direction parallel to the tissue surface and at an angle <15°, pushing forward, backward, and to the sides in small amplitude. Along the gaps between the tissues, the lifting and pushing operations are alternately performed to peel off the submucosa and mucosa (or to obtain a basement membrane complex layer containing part of the submucosa).

[0062] (3) Gradient decellularization: The basement membrane composite layer obtained in step (2) was immersed in 7 mM Tris-HCl (pH 8.0) and shaken on a constant temperature shaker at 4°C for 16 h. After shaking, it was rinsed 5 times with sterile PBS. Then, it was shaken at room temperature for 28 h in 1.2% sodium deoxycholate (containing 1 mM MPMSF). After shaking, it was rinsed 5 times with sterile PBS until the rinsing solution was clear. It was then transferred to sterile 0.1 M Tris-HCl (pH 8.0) containing DNase I (50 U / mL) and RNase A (50 U / mL) and shaken at 37°C for 3.5 h. After treatment, it was rinsed 5 times with sterile PBS and then rinsed with sterile ultrapure water until the rinsing solution was free of foam.

[0063] (4) Freeze-drying: The matrix (the substance treated in step (3)) is spread in a single layer on a freeze-drying tray and pre-frozen at -75℃ for 4.5h. Primary drying: -45℃, vacuum degree 4Pa, for 18h; secondary drying: 27℃, vacuum degree 2Pa, for 8h.

[0064] (5) Pathogen inactivation: Cobalt-60 γ-ray irradiation was used with an irradiation dose of 10.0 kGy.

[0065] (6) Cryogenic grinding and grading: Under aseptic conditions, a mortar pre-cooled by liquid nitrogen for 30 min was placed in a sterile laminar flow hood, and an irradiated lyophilized basement membrane was added, with liquid nitrogen continuously replenished. The membrane was ground into powder using a sterile pestle, with liquid nitrogen replenished every 30 s during the grinding process. The membrane was then sieved sequentially to collect the basement membrane particles, which were then placed into sterile centrifuge tubes for later use.

[0066] Preparation Example 5: Preparation of Basement Membrane Microparticles Compared with Preparation Example 2, the raw material was placental amnion, and all other conditions were the same.

[0067] II. Examples of Tissue Regeneration Fillers Example 1 Take 1.8 g of decellularized matrix microparticles (particle size 150-300 μm) prepared above and 0.18 g of basement membrane microparticles (particle size 50-100 μm) prepared in Preparation Example 1, add them to a dispersion of 1.4 g of glycerol and 0.5 g of polysorbate 60, and stir thoroughly to obtain a dispersion phase without white hard lumps.

[0068] Weigh 2.0 g of sodium hyaluronate and add it to 65.0 g of phosphate-sodium chloride buffer (the mass ratio of sodium chloride to disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH=7.0) in the phosphate-sodium chloride buffer is 2:63). Stir at 40°C until completely dissolved, then add 0.05 g of amino acids and mix well to obtain the non-crosslinked gel matrix.

[0069] The dispersed phase was added to the non-crosslinked gel matrix, and the above phosphate-sodium chloride buffer was added to make up to 100.0g. After mixing evenly in a vacuum stirrer, it was aseptically filled into pre-filled syringes to obtain the final product.

[0070] Example 2: Take 1.5 g of decellularized matrix microparticles (particle size 25-50 μm) prepared above and 0.5 g of basement membrane microparticles (particle size 1-25 μm) prepared in Preparation Example 2, add them to a dispersion of 1.0 g of glycerol and 0.5 g of polysorbate 60, and stir thoroughly to obtain a dispersion phase without white lumps.

[0071] Weigh 2.0 g of cross-linked sodium hyaluronate and add it to 80.0 g of hydroxyethylpiperazine ethanesulfonic acid-trehalose buffer (where the mass ratio of trehalose to hydroxyethylpiperazine ethanesulfonic acid buffer (pH=7.0) in the hydroxyethylpiperazine ethanesulfonic acid-trehalose buffer is 2:78). Stir at 40°C until completely dissolved, then add 0.3 g of lidocaine hydrochloride and mix well to obtain the cross-linked gel matrix.

[0072] The dispersed phase was added to the cross-linked gel matrix, and the above-mentioned hydroxyethylpiperazine ethanesulfonic acid-trehalose buffer was added to make up to 100.0 g. After mixing evenly in a vacuum stirrer, it was aseptically filled into pre-filled syringes to obtain the final product.

[0073] Example 3: Take 1.0 g of decellularized matrix microparticles (particle size 25-50 μm) prepared in Preparation Example 1 and 1.0 g of basement membrane microparticles (particle size 1-25 μm) prepared in Preparation Example 1, add them to 2.0 g of glycerol, and stir thoroughly to obtain a dispersed phase without white lumps.

[0074] Weigh 1.0 g of sodium hyaluronate and add it to 65.0 g of phosphate-sodium chloride buffer (the mass ratio of sodium chloride to disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH=7.0) in the phosphate-sodium chloride buffer is 2:63). Stir at 40°C until completely dissolved, then add 0.1 g of amino acids and mix well to obtain the non-crosslinked gel matrix.

[0075] The dispersed phase was added to the non-crosslinked gel matrix, and the above-mentioned phosphate-sodium chloride buffer was added to make up to 100.0g. After being mixed evenly in a vacuum stirrer, it was aseptically filled into vials to obtain the final product.

[0076] Example 4: Take 1.5 g of decellularized matrix microparticles (particle size 25-50 μm) prepared in Preparation Example 1 and 0.3 g of basement membrane microparticles (particle size 1-25 μm) prepared in Preparation Example 4, add them to a dispersion of 5.0 g of physiological saline, and stir thoroughly to obtain a dispersion phase without white lumps.

[0077] Weigh 2.0 g of cross-linked sodium hyaluronate and add it to 80.0 g of hydroxyethylpiperazine ethanesulfonic acid-trehalose buffer (where the mass ratio of trehalose to hydroxyethylpiperazine ethanesulfonic acid buffer (pH=7.0) in the hydroxyethylpiperazine ethanesulfonic acid-trehalose buffer is 2:78). Stir at 40°C until completely dissolved, then add 0.3 g of lidocaine hydrochloride and 0.05 g of amino acids, and mix well to obtain the cross-linked gel matrix.

[0078] The dispersed phase was added to the cross-linked gel matrix, and the above-mentioned hydroxyethylpiperazine ethanesulfonic acid-trehalose buffer was added to make up to 100.0 g. After being mixed evenly in a vacuum stirrer, the mixture was aseptically filled into vials and freeze-dried.

[0079] Example 5: Compared with Example 2, 0.5 g of basement membrane microparticles (particle size 1-25 μm) prepared in Preparation Example 5 were selected, and all other conditions were the same.

[0080] Example 6: Take 2.4 g of decellularized matrix microparticles (particle size 25-50 μm) prepared above and 0.6 g of basement membrane microparticles (particle size 1-25 μm) prepared in Preparation Example 3, add them to a dispersion of 1.5 g of glycerol and 0.8 g of polysorbate 60, and stir thoroughly to obtain a dispersion phase without white lumps.

[0081] Weigh 2.5 g of cross-linked sodium hyaluronate and add it to 78.0 g of hydroxyethylpiperazine ethanesulfonic acid-trehalose buffer (where the mass ratio of trehalose to hydroxyethylpiperazine ethanesulfonic acid buffer (pH=7.0) in the hydroxyethylpiperazine ethanesulfonic acid-trehalose buffer is 3:75). Stir at 40°C until completely dissolved, then add 0.2 g of lidocaine hydrochloride and mix well to obtain the cross-linked gel matrix.

[0082] The dispersed phase was added to the cross-linked gel matrix, and the above-mentioned hydroxyethylpiperazine ethanesulfonic acid-trehalose buffer was added to make up to 100.0 g. After being mixed evenly in a vacuum stirrer, it was aseptically filled into pre-filled syringes to obtain the final product.

[0083] Example 7: Take 4.2 g of decellularized matrix microparticles (particle size 25-50 μm) prepared above and 0.6 g of basement membrane microparticles (particle size 1-25 μm) prepared in Preparation Example 4, add them to a dispersion of 2.0 g of glycerol and 0.6 g of polysorbate 60, and stir thoroughly to obtain a dispersion phase without white lumps.

[0084] Weigh 3.0 g of cross-linked sodium hyaluronate and add it to 75.0 g of hydroxyethylpiperazine ethanesulfonic acid-trehalose buffer (where the mass ratio of trehalose to hydroxyethylpiperazine ethanesulfonic acid buffer (pH=7.0) in the hydroxyethylpiperazine ethanesulfonic acid-trehalose buffer is 1:74). Stir at 40°C until completely dissolved, then add 0.4 g of lidocaine hydrochloride and mix well to obtain the cross-linked gel matrix.

[0085] The dispersed phase was added to the cross-linked gel matrix, and the above-mentioned hydroxyethylpiperazine ethanesulfonic acid-trehalose buffer was added to make up to 100.0 g. After being mixed evenly in a vacuum stirrer, it was aseptically filled into pre-filled syringes to obtain the final product.

[0086] Example 8: Take 4.0 g of decellularized matrix microparticles (particle size 25-50 μm) prepared above and 0.5 g of basement membrane microparticles (particle size 1-25 μm) prepared in Preparation Example 5, add them to a dispersion of 1.2 g of glycerol and 0.7 g of polysorbate 60, and stir thoroughly to obtain a dispersion phase without white lumps.

[0087] Weigh 2.2 g of cross-linked sodium hyaluronate and add it to 79.0 g of hydroxyethylpiperazine ethanesulfonic acid-trehalose buffer (where the mass ratio of trehalose to hydroxyethylpiperazine ethanesulfonic acid buffer (pH=7.0) in the hydroxyethylpiperazine ethanesulfonic acid-trehalose buffer is 5:74). Stir at 40°C until completely dissolved, then add 0.35 g of lidocaine hydrochloride and mix well to obtain the cross-linked gel matrix.

[0088] The dispersed phase was added to the cross-linked gel matrix, and the above-mentioned hydroxyethylpiperazine ethanesulfonic acid-trehalose buffer was added to make up to 100.0 g. After being mixed evenly in a vacuum stirrer, it was aseptically filled into pre-filled syringes to obtain the final product.

[0089] Comparative Example 1 Compared to Example 2, this example did not contain basement membrane microparticles, and all other conditions were the same.

[0090] Comparative Example 2 Compared to Example 2, this example did not contain decellularized matrix microparticles, but all other conditions were the same.

[0091] Comparative Example 3 Commercially available cross-linked sodium hyaluronate gel.

[0092] III. Experimental Examples 1. Physicochemical property testing of tissue regeneration fillers pH, osmotic pressure concentration, elastic modulus, and extrusion force were measured for Examples 1-5 respectively, and the specific procedures are as follows: Sample preparation: Examples 1, 2, and 5 were measured directly; Example 3 was slowly drawn out with a syringe before use to avoid air bubbles; Example 4 was reconstituted with 1.0 mL of sterile water for injection and then slowly drawn out with a syringe.

[0093] pH: Determined according to the method specified in General Chapter 0631, Part IV, 2025 edition of the Pharmacopoeia of the People's Republic of China.

[0094] Osmotic pressure: The osmotic concentration of the sample was measured using freezing point osmotic pressure (OM 819°C). The specific operating procedure followed the method specified in Section 0632, General Chapter IV, of the 2025 edition of the Pharmacopoeia of the People's Republic of China, for the determination of osmolar concentration.

[0095] Pushing force: Examples 1, 2, and 5 are equipped with 27 G needles, and Examples 3 and 4 are equipped with 30 G needles, and are pushed at a speed of 10 mm / min.

[0096] Elastic modulus: The elastic modulus (G') at 1 Hz is measured using a rheometer at a shear strain of 1 and a temperature of 25°C, based on a frequency scan (0.01~5Hz), as well as the ratio of viscosity to elasticity (Tanδ=G'' / G').

[0097] like Figure 1 As shown, the liquid tissue regeneration filler has a uniform milky white appearance, while the freeze-dried tissue regeneration filler has a uniform sponge-like texture. Furthermore, based on the basic physicochemical properties of the tissue regeneration filler, as shown in Table 1, the results indicate that it possesses good injectability and biocompatibility.

[0098] Table 1. Test results of basic physicochemical properties of tissue regeneration fillers

[0099] 2. Animal experiments: subcutaneous injection promotes the formation of the basement membrane and extracellular matrix. Sample preparation: After installing 27G needles, the samples from Example 2, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were ready for use.

[0100] Experimental animals (SD rats) were anesthetized with pentobarbital via intraperitoneal injection, and the skin on their backs was disinfected with iodine and ethanol. The above-mentioned sample was slowly injected into the subcutaneous tissue on the back of the rats at two sites, 500 μL at each site, for a total injection of 1.0 mL per rat. On day 28 post-injection, the animals were randomly divided into two batches for RNA extraction and reverse transcription of cDNA. The gene expression levels of type IV collagen (Col4a1), laminin (Lamb1), and type I collagen (Col1a1) in each group were then detected by RT-qPCR. Figure 2As shown, the tissue regeneration filler prepared in this invention can significantly increase the gene expression level of major extracellular matrix proteins such as the body's own basement membrane and collagen after subcutaneous injection.

[0101] 3. Animal experiments: Subcutaneous injection promotes the formation of the basement membrane and extracellular matrix. Sample preparation: Examples 1, 2, and 5 can be used directly after installing a 27G needle. Example 3 requires slow withdrawal with an injection needle before use to avoid air bubbles. Example 4 requires reconstitution with 1.0 mL of sterile water for injection, followed by repeated shaking and slow withdrawal with an injection needle for later use.

[0102] Experimental animals (SD rats) were anesthetized with pentobarbital via intraperitoneal injection, and the skin on their backs was disinfected with iodine and ethanol. The above-mentioned sample was slowly injected into the subcutaneous tissue on the back of the rats at two sites, 500 μL at each site, for a total injection of 1.0 mL per rat. At 1 month and 6 months post-injection, samples were randomly collected from each group in two batches for RNA extraction and reverse transcription of cDNA. The gene expression levels of type IV collagen (Col4a1), laminin (Lamb1), and type I collagen (Col1a1) in each group were then detected by RT-qPCR. Figure 3 As shown, the tissue regeneration filler prepared by this invention can prolong the gene expression levels of major extracellular matrix proteins such as the body's own basement membrane and collagen. Figure 4 As shown, further research indicates that once the material is completely degraded in vivo, and after an observation period sufficient to reflect its long-term biological effects, it no longer has a sustained promoting effect on the gene expression of Col4a1, Lamb1, and Col1a1. Specifically, with the degradation of the material, the gene expression levels of these proteins can return to baseline, and no delayed or recurrent upregulation effect was observed. This confirms that the material does not continuously interfere with the body's normal matrix metabolism processes after complete degradation, demonstrating clear safety and controllable characteristics.

[0103] 4. Animal experiments: Regulation of the dynamic microenvironment after subcutaneous injection Sample preparation: Examples 1, 2, 5, Comparative Examples 1, 2, and 3 were used directly after installing 27G needles. For Example 3, the sample was slowly withdrawn with an injection needle before use to avoid air bubbles. For Example 4, the sample was reconstituted with 1.0 mL of sterile water for injection, shaken repeatedly, and then slowly withdrawn with an injection needle for later use.

[0104] Experimental animals (SD rats) were anesthetized with pentobarbital via intraperitoneal injection, and the skin on their backs was disinfected with iodine and ethanol. The above-mentioned sample was slowly injected into the subcutaneous tissue on the back of the rats at two sites, 500 μL at each site, for a total injection of 1.0 mL per rat. On day 7 post-injection, the animals were randomly divided into two batches for RNA extraction and reverse transcription of cDNA. The gene expression levels of transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) in each group were then detected by RT-qPCR. Figure 5 As shown, the tissue regeneration filler prepared in this invention can significantly upregulate the mRNA expression of TGF-β1 and VEGF in fibroblasts after subcutaneous injection, indicating that the tissue regeneration filler can initiate tissue synthesis and vascularization processes and regulate matrix remodeling, thereby creating a dynamic microenvironment conducive to high-quality, functional tissue regeneration.

[0105] 5. Animal experiments: Subcutaneous injection of type IV collagen-specific staining test Example 2: An animal model was established by intradermal injection into the back of SD rats. Tissue samples were collected on day 7 post-implantation. The tissue blocks were fixed in 4% paraformaldehyde, embedded in paraffin, and then sectioned to a thickness of 4 μm. Immunohistochemistry was used for type IV collagen specificity detection. After antigen retrieval with sodium citrate buffer, overnight incubation with rabbit anti-rat type IV collagen primary antibody at 4°C, and room temperature incubation with HRP-labeled secondary antibody, the samples were developed using DAB staining and counterstained with hematoxylin. Figure 6 As shown, under an optical microscope, abundant brown granules were observed in the cytoplasm of fibroblasts in the implantation area, and the newly formed basement membrane-like structures all showed obvious type IV collagen positive signals, confirming that the material can effectively induce host cells to synthesize characteristic components of the basement membrane and participate in functional structural reconstruction.

[0106] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A tissue regeneration filler, characterized in that, The components include the following parts by weight: 1.0 to 5.0 parts of mixed microparticles; The mixed microparticles comprise decellularized matrix microparticles and basement membrane microparticles, wherein the mass ratio of decellularized matrix microparticles to basement membrane microparticles is 10⁻¹:

1.

2. The tissue regeneration filler as described in claim 1, characterized in that, The particle size range of the decellularized matrix microparticles is 10 μm to 300 μm; the particle size range of the basement membrane microparticles is 1 to 100 μm.

3. The tissue regeneration filler as described in claim 1, characterized in that, The mass ratio of the decellularized matrix microparticles to the basement membrane microparticles is 5-3:1; The decellularized matrix microparticles are selected from one or more of the following: skin, small intestine, diaphragm, meninges, pericardium, fascia, nerve, kidney, placenta, bladder, blood vessels, greater omentum, tendon, and muscle, with the small intestine being preferred.

4. The tissue regeneration filler as described in claim 1, characterized in that, The tissue regeneration filler also includes the following components in parts by weight: 1.0-8.0 parts dispersion, 0.5-8.0 parts osmotic pressure regulator, 60.0-95.0 parts pH buffer, and 1.0-30.0 parts gel forming agent; The dispersion is selected from at least one of physiological saline, glycerol, propylene glycol, polyethylene glycol, Tween 80, and polysorbate 60; The osmotic pressure regulator is selected from one or more of trehalose, mannitol, sorbitol, sodium chloride, and sucrose; The pH buffer solution is selected from at least one of the following: acetate-sodium acetate buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer, citrate-sodium citrate buffer, and hydroxyethylpiperazine ethanesulfonic acid buffer. The gelling agent is selected from one or more of sodium hyaluronate, cross-linked sodium hyaluronate, methylcellulose, collagen, povidone, chitosan, agarose, and sodium alginate.

5. The tissue regeneration filler as described in claim 1, characterized in that, The tissue regeneration filler also contains one or more of an anesthetic and a nutrient, wherein the anesthetic is present in a mass fraction of 0.05 to 1 part and the nutrient is present in a mass fraction of 0.01 to 1 part. The anesthetic agent is selected from any one of lidocaine hydrochloride, prilocaine, procaine, benzocaine, bupivacaine, and tetracaine; The nutrients are selected from one or more of the following: amino acids, vitamins, polynucleotides, polynucleotides, coenzymes, minerals, trace elements, nucleic acids, glutathione, and polypeptides.

6. A method for preparing the tissue regeneration filler according to any one of claims 1-5, characterized in that, Includes the following steps: S1. Basement membrane microparticles were prepared by sequentially pretreating biological tissues, mechanically separating them, decellularizing them, freeze-drying them, inactivating pathogens, and grinding them at ultra-low temperatures. S2. Mix the basement membrane microparticles obtained in step S1 with the decellularized matrix microparticles and dispersion to form a dispersed phase; S3. Mix the gel forming agent, osmotic pressure regulator and pH buffer solution, stir well, and prepare the gel matrix; Preferably, in step S3, after stirring evenly, one or more of the anesthetic and nutrient agents are added and mixed evenly. S4. The dispersed phase obtained in step S2 is mixed with the gel matrix to obtain a tissue regeneration filler.

7. The preparation method according to claim 6, characterized in that, In step S1, the method for preparing basement membrane microparticles includes the following steps: (1) Mechanical layering: The pretreated biological tissue is sequentially peeled off the serosa and outer muscle layer, and the mucosal surface of the remaining tissue is treated with collagenase and trypsin. After treatment, it is rinsed and then peeled off to obtain the target tissue layer. (2) Gradient decellularization: The target tissue layer was successively soaked and shaken in Tris-HCl solution, then in sodium deoxycholate solution containing PMSF, and then in a mixture of Tris-HCl containing DNase I and RNase A. After each shaking treatment, the tissue was rinsed. (3) Post-processing: The decellularized target tissue layer was freeze-dried, irradiated with gamma rays, and ground and sieved in liquid nitrogen.

8. The preparation method according to claim 7, characterized in that, In step (1), the pretreatment is as follows: the biological tissue is squeezed and rinsed 3-7 times in sterile physiological saline, transferred to PBS containing 0.1-0.2 mg / mL amoxicillin and 0.05-0.1 mg / mL gentamicin, and stored at 4-6℃ for 12-24h, with the antibiotic solution being replaced every 6h; In step (1), the biological tissue is selected from one or more of the following: bladder, retina, placental amnion, corneal posterior elastic layer, cerebral blood vessels, and renal tubules, preferably the bladder; In step (1), the concentration of collagenase is 0.01%-0.1%, the concentration of trypsin is 0.01%-0.5%, and the enzyme treatment time is 5-15 min.

9. The preparation method according to claim 7, characterized in that, In step (2), the solution was shaken at 3-5°C for 12-24 h in 5-10 mM Tris-HCl solution; shaken at room temperature for 24-36 h in 0.5-2% sodium deoxycholate solution containing 1 mM PMSF; shaken at 35-40°C for 2-4 h in a Tris-HCl mixture containing 50 U / mL DNase I and 50 U / mL RNase A; after each shaking treatment, the solution was rinsed with PBS 4-7 times, and after the last shaking treatment, it was rinsed with ultrapure water until no foam was present. In step (3), the freeze drying is performed as follows: first, pre-freezing at -60~-80℃ for 4~6h, then primary drying at -40~-60℃ for 12~24h under vacuum conditions <10Pa, and secondary drying at 25~30℃ for 6~12h; the dose of the γ-ray irradiation is 8~15 kGy.

10. The application of a tissue regeneration filler as described in any one of claims 1-5 or a tissue regeneration filler prepared by the preparation method described in any one of claims 6-8 in the preparation of biomedical materials or medical aesthetic materials; Preferably, the dosage form of the material is a gel or a lyophilized agent, and more preferably a gel.

Images