An angiogenic filling repair agent and a method for preparing the same

Abstract

The application discloses a kind of proangiogenic filling repair agent and its preparation method, the filling repair agent is composed of vascular endothelial cell supernatant, acellular matrix microparticle, suspending agent and protective agent, vascular endothelial cell supernatant is collected cell culture fluid after being induced into tube under low oxygen three-dimensional condition by vascular endothelial cell, and is concentrated after centrifugation;Acellular matrix microparticle is obtained by collecting after tissue matrix is acellular, shearing, freeze-drying, liquid nitrogen grinding;The proangiogenic filling repair agent is obtained by mixing the above two with suspending agent, protective agent, the application combines vascular endothelial cell supernatant with acellular matrix microparticle, contains high concentration angiogenesis-related factor and stable three-dimensional biomimetic scaffold structure, realizes functional complementation by the synergistic effect of "biological active factor + biomimetic scaffold", so as to innovatively obtain a kind of filling repair agent which realizes promoting angiogenesis, constructs physical support architecture, promotes tissue repair regeneration ability simultaneously.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a filler repair agent that promotes angiogenesis and its preparation method. Background Technology

[0002] In clinical treatment, chronic wounds (such as diabetic foot ulcers, pressure ulcers, and chronic burn wounds), traumatic soft tissue defects (including large-area skin avulsions, tissue loss due to war trauma, and tissue necrosis after severe crush injuries), congenital tissue malformations (such as soft tissue defects in cleft lip and palate, and congenital skin dysplasia), as well as bone nonunion after bone trauma and bone defects caused by osteomyelitis, all face the dual challenges of structural repair and blood supply reconstruction. During the development of these diseases, local blood circulation disorders and collagen loss coexist. While simply supplementing collagen can fill the defects, it is difficult to solve the problem of insufficient blood supply. Traditional treatments often address one aspect while neglecting another. Therefore, novel treatment plans that combine filling and support functions with angiogenesis promotion have become an urgent clinical need. Thus, there is an urgent need to develop an angiogenesis-promoting filling and repair agent and its preparation method to solve the above-mentioned technical problems.

[0003] In view of this, the present invention is hereby proposed. Summary of the Invention

[0004] The purpose of this invention is to provide a pro-angiogenic filler and repair agent and its preparation method. By combining vascular endothelial cell supernatant with decellularized matrix microparticles, and through the synergistic effect of "bioactive factors + biomimetic scaffold", complementary functions are achieved. That is, it contains a high concentration of angiogenesis-related factors and has a three-dimensional biomimetic scaffold structure of decellularized matrix microparticles, thereby obtaining a filler and repair agent that can promote angiogenesis, build a physical support structure, and promote tissue repair and regeneration. This filler and repair agent has broad application prospects and is conducive to its widespread application.

[0005] To achieve the above objectives, the present invention provides a method for preparing an angiogenesis-promoting filler repair agent, comprising the following steps:

[0006] S1: Passage vascular endothelial cells cultured to generation P2-P5 onto matrix gel, and culture them statically under hypoxic conditions to induce vascular endothelial cells to form tubes. Observe under a microscope, and collect the cell culture medium when more than 80% of the cells have formed tubular branching structures.

[0007] S2: After centrifuging the cell culture medium collected in S1 at ultra-high speed, collect the supernatant and concentrate the supernatant;

[0008] S3: Cut the decellularized matrix into small pieces, freeze-dry them, grind them in liquid nitrogen, and collect the decellularized matrix particles after the liquid nitrogen has completely evaporated.

[0009] S4: Mix the supernatant after S2 concentration with the decellularized matrix microparticles collected in S3, add suspending agent and protectant, and homogenize to obtain an angiogenesis-promoting filling and repair agent.

[0010] Preferably, in S1, the vascular endothelial cells are one of the following: human umbilical vein endothelial cells (HUVECs), hematopoietic endothelial cells (CD32-positive endothelial cells), endothelial progenitor cells (EPCs), or human iPS cells induced to differentiate into vascular endothelial cells; the matrix gel is a soluble basement membrane extract of EHS mouse sarcoma, with a matrix concentration of not less than 8 mg / mL; the specific method for matrix gel plating is as follows: the matrix gel is thawed at 4°C overnight, 0.5 mL of matrix gel is placed in a culture flask pre-cooled at 4°C, incubated at 4°C for 30 min, then transferred to 37°C and incubated horizontally for 0.5-2 h until the matrix gel is completely solidified, providing a three-dimensional culture environment for the formation of vascular endothelial cells; the culture medium used for culturing vascular endothelial cells is ECM-specific medium, with fetal bovine serum added at a concentration of 0.01-0.5%.

[0011] Preferably, in step S1, the specific method for inducing vascular endothelial cells to form tubes is as follows: resuspending vascular endothelial cells in culture medium, counting them, and then preparing a solution of 0.5-2 × 10⁻⁶ cells / mL. 5 Add cell suspension to a matrix gel at a density of 5000-20000 cells / cm³. 2 After gently tapping and shaking to mix, place in a low-oxygen incubator (temperature 37℃, CO2 concentration 5%, O2 concentration 5-10%) and incubate for 2-6 hours. Observe every 1 hour during this period. Under a microscope, observe that more than 80% of the cells have elongated into tubular branches and the cells are connected to form a network structure.

[0012] Preferably, in step S2, the specific method for collecting the supernatant is as follows: The vascular endothelial cell culture supernatant is aspirated into a centrifuge tube and centrifuged at 100,000-500,000g at 4°C for 1-3 hours to collect the supernatant. The specific method for concentrating the supernatant is as follows: The supernatant is transferred into a concentration tube containing a 30kDa filter membrane and centrifuged at 3000g at 4°C for 10 minutes. After mixing by pipetting, the mixture is centrifuged again. This process is repeated until the concentration is stopped when the VEGF (vascular endothelial growth factor) content in the solution is higher than 5 ng / mL and the Ang-1 (angiogenic factor 1) content is higher than 0.5 ng / mL, as detected by ELISA.

[0013] Preferably, in step S3, the decellularized matrix is ​​prepared from allogeneic or xenogeneic tissues, including human, bovine, ovine, swine, or equine tissues. The tissues include dermis, bladder, submucosa of small intestine, tendon, placenta, amnion, or umbilical cord. The specific method for decellularizing the tissue matrix is ​​as follows: after washing the fresh tissue, hair and fat layer are removed. The tissue is then mixed at 37°C with 0.25-1% trypsin solution, 70% isopropanol containing 0.1-0.5% SDS, 70% isopropanol containing 1-2% Triton X-100, and isopropanol. The solution is changed each time and the tissue is ultrasonically washed 5 times with purified water to remove residual reagents.

[0014] Preferably, in step S3, the specific method for freeze-drying is as follows: the decellularized matrix is ​​cut into pieces smaller than 1 cm. 2 Small pieces of the decellularized matrix were spread flat in a freeze-drying tray and frozen at -40℃ for 6-24 h. Then, they were transferred to a freeze dryer with the temperature set to -40℃ and the vacuum set to 10-100 Pa for 12-48 h. The specific method for grinding in liquid nitrogen was as follows: the freeze-dried decellularized matrix pieces were placed in a mortar and pestle, and liquid nitrogen was poured in until the matrix was completely submerged. The matrix was gently ground in a circular motion with a pestle for 0.5-2 h. During this period, liquid nitrogen was added to ensure that the sample was submerged. After the temperature was raised to 4℃, the liquid nitrogen was allowed to evaporate completely, and the decellularized matrix particles were collected.

[0015] Preferably, in step S4, the suspending agent is one or two of sodium hyaluronate, methylcellulose, carboxymethyl chitosan, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, carbomer, polyvinyl alcohol, poloxamer, gelatin, collagen, and silk fibroin. The concentration of the suspending agent is such that the viscosity of the suspending agent solution is between 300-1000 mPa·s. The protective agent is one or two of sucrose, trehalose, mannitol, glycerol, glycine, arginine, and polyethylene glycol.

[0016] Preferably, in step S4, the concentrated vascular endothelial cell supernatant and decellularized matrix microparticles are mixed at a ratio of (10-30):1 (v / w), and after adding a suspending agent and a protective agent, the mixture is homogenized at 4°C for 0.5-2 h. The mixed paste is collected, poured into a container, and the liquid is sealed in a pre-filled syringe using a filling machine. After irradiation sterilization, it is used for injection.

[0017] The present invention also provides a filler for promoting angiogenesis prepared by the above-described method for preparing an angiogenesis-promoting filler for repair.

[0018] The present invention provides a filler and repair agent that promotes angiogenesis and its preparation method, which has the following beneficial effects.

[0019] 1. This invention combines vascular endothelial cell supernatant with decellularized matrix microparticles to achieve functional complementarity. The decellularized matrix, derived from natural tissue, retains a three-dimensional biomimetic scaffold composed of collagen fibers, elastin, and glycosaminoglycans. This scaffold precisely fills soft tissue or bone defects, providing mechanical support and guiding cell adhesion. The vascular endothelial cell supernatant is enriched with angiogenesis-promoting factors, which continuously release active substances after implantation, inducing the directional migration of host vascular endothelial cells to penetrate the scaffold pores and form new blood vessels. This synergistic effect of "bioactive factors + biomimetic scaffold" effectively solves the contradiction between insufficient blood supply and lack of support in traditional materials, providing an innovative solution for tissue repair that combines structure and function. It is expected to significantly improve the treatment effect of clinical challenges such as tissue defects and bone repair, opening up new pathways in the field of regenerative medicine.

[0020] 2. Compared with traditional collagen filling and repair agents, this invention combines vascular endothelial cell supernatant with decellularized matrix microparticles to simultaneously provide damaged tissue with bioactive factors that promote angiogenesis and a three-dimensional collagen scaffold. This dual-function synergistic design breaks through the limitations of single materials and demonstrates superior therapeutic efficacy in accelerating blood revascularization and promoting tissue repair.

[0021] 3. Compared to ordinary cell supernatant, this invention involves first culturing vascular endothelial cells on a matrix gel and then stimulating them under hypoxic conditions. This activates the endothelial cells, causing their morphology to change from polygonal to tubular branching, significantly increasing the secretion of angiogenesis-related active factors. The cell culture supernatant is then collected. Furthermore, the concentrated supernatant increases factor concentration while removing small molecule impurities. Tissue repair relies on adequate blood supply. A large amount of angiogenesis-related growth factors and bioactive substances stimulate the proliferation and migration of vascular endothelial cells within the tissue, promoting the formation of new blood vessels and improving blood supply to damaged tissue, thereby promoting tissue repair.

[0022] 4. Compared to traditional collagen-filled repair agents, this invention uses microparticles prepared from decellularized matrix. These microparticles remove immunogenic cellular components while retaining the three-dimensional structure and bioactive components of the extracellular matrix. Compared to extracted, processed, or in vitro expressed collagen, the decellularized matrix provides a physical support closer to the in vivo environment for cell adhesion, proliferation, and differentiation. Furthermore, in addition to collagen, the decellularized matrix contains various bioactive components, such as elastic fibers, glycosaminoglycans, and growth factors. These components work together to regulate cell behavior and promote tissue repair and regeneration. The decellularized matrix retains the collagen fiber scaffold and basic tissue structure, providing an environment similar to the extracellular matrix for damaged tissues, which helps cell adhesion, proliferation, and differentiation, promoting the repair and regeneration of damaged tissues. Decellularized matrix is ​​derived from human or animal tissues and, after processing, removes cellular components, reducing immunogenicity. It has good biocompatibility, is less likely to cause significant immune rejection, and can function relatively stably in vivo, providing favorable conditions for tissue repair and functional improvement.

[0023] 5. The components and contents of this invention are the optimal ratio obtained through scientific research and repeated screening. The vascular endothelial cell supernatant and decellularized matrix microparticles are mixed at a ratio of (10-30):1 (v / w), achieving both the fluidity of the filler repair agent and the stability of the microparticles. Furthermore, the viscosity of the suspending agent solution should be controlled between 300-1000 mPa·s, providing support for the decellularized matrix microparticles while avoiding excessively viscous solutions that would cause injection difficulties. This invention achieves a harmonious balance between the filler repair agent's biological activity, formulation stability, material mechanical properties, and ease of operation, providing a safe and efficient new solution for tissue damage repair and treatment, with significant clinical translational value and broad application prospects. Attached Figure Description

[0024] Figure 1 This is a particle size distribution diagram of decellularized matrix particles;

[0025] Figure 2 Scanning electron microscope image of the filling repair agent;

[0026] Figure 3 The rate at which the filler degrades over time;

[0027] Figure 4 The extrusion force variation curves of the filling repair agent (A is the extrusion force curve of Example 1, and B is the extrusion force curve of Comparative Example 3).

[0028] Figure 5 The filling repair agent forms are shown in Figure A (Form 1) and Figure B (Form 4). Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments and accompanying drawings to help understand the content of the present invention. Example 1

[0030] The raw materials for the preparation of Example 1 are human umbilical vein endothelial cells and bovine decellularized dermal matrix. The specific steps are as follows:

[0031] (1) Human umbilical vein endothelial cells cultured to P3 generation were passaged onto matrix gel and cultured statically in a 37℃, 5% CO2, 8% O2 incubator for 4 h. Under a microscope, most cells formed tubular branching structures, and the cell culture medium was collected.

[0032] (2) The collected culture medium was centrifuged at 4°C at ultra-high speed (300,000g) and the supernatant was collected. The supernatant was concentrated using a 30 kDa filter membrane. The VEGF (vascular endothelial growth factor) content in the solution was detected by ELISA to be 75.2 ng / mL and the Ang-1 (angiopoietin 1) content was 25.9 ng / mL.

[0033] (3) Cut the decellularized bovine hide matrix into pieces smaller than 1 cm. 2 Small pieces of decellularized matrix were freeze-dried at -40℃ for 30 h; small pieces of decellularized matrix were immersed in liquid nitrogen, ground for 2 h, and then heated to 4℃ until the liquid nitrogen completely evaporated to obtain decellularized matrix microparticles.

[0034] (4) Add the components according to Table 1:

[0035] Table 1 Component Addition Amount

[0036]

[0037] Weigh the above components according to Table 1, mix them using a homogenizer, and then fill them into syringes to obtain the finished filling and repair agent.

[0038] Particle size distribution experiment:

[0039] The decellularized matrix microparticle powder prepared by liquid nitrogen grinding in Example 1 was analyzed for particle size distribution using a laser particle size analyzer. The results are as follows: Figure 1 As shown, the particle size of the decellularized matrix microparticles is mainly in the range of 150-550 μm, with a volume average particle size D[4,3] of 275.213 μm, and the particle size distribution is concentrated.

[0040] Scanning electron microscopy observation of particle morphology:

[0041] Take 0.1g of Example 1, gently coat it into a 24-well plate, freeze-dry overnight, remove a small piece of freeze-dried sample and attach it to the stage of a scanning electron microscope, sputter-coat it with gold and observe the sample morphology. Figure 2As shown, the freeze-dried filler repair agent contains a large amount of collagen fibers, forming a three-dimensional network structure with pores, which facilitates the growth of fibroblasts in vivo. Example 2

[0042] The raw materials for the preparation of Example 2 were vascular endothelial progenitor cells and porcine decellularized dermal matrix. The specific steps were as follows:

[0043] (1) The vascular endothelial progenitor cells cultured to the P2 generation were passaged onto a matrix gel and cultured statically for 6 h in a 37℃, 5% CO2, 10% O2 incubator. Under a microscope, most of the cells formed tubular branching structures, and the cell culture medium was collected.

[0044] (2) After centrifuging the collected culture medium at 4℃ for 2 h at ultra-high speed (300,000g), the supernatant was collected. The supernatant was concentrated using a 30kDa filter membrane. The VEGF (vascular endothelial growth factor) content in the solution was detected by ELISA to be 136.8 ng / mL and the Ang-1 (angiopoietin 1) content was 43.4 ng / mL.

[0045] (3) Cut the decellularized pig skin matrix into pieces smaller than 1 cm. 2 Small pieces of the decellularized matrix were freeze-dried at -40℃ for 30 h; the small pieces of decellularized matrix were immersed in liquid nitrogen, ground for 1 h, and then heated to 4℃ until the liquid nitrogen completely evaporated to obtain decellularized matrix microparticles.

[0046] (4) Add the components according to Table 2:

[0047] Table 2 Component Addition Amount

[0048]

[0049] Weigh the above components according to Table 2, mix them using a homogenizer, and then fill them into syringes to obtain the finished filling and repair agent. Example 3

[0050] The raw materials for the preparation of Example 3 were vascular endothelial progenitor cells and bovine Achilles tendon. The specific steps were as follows:

[0051] (1) The vascular endothelial progenitor cells cultured to generation P4 were passaged onto a matrix gel and cultured statically for 6 h in a 37°C, 5% CO2, 6% O2 incubator. Under a microscope, most of the cells formed tubular branching structures, and the cell culture medium was collected.

[0052] (2) After centrifuging the collected culture medium at 4℃ for 1 h at ultra-high speed (500,000g), the supernatant was collected. The supernatant was concentrated using a 30 kDa filter membrane. The VEGF (vascular endothelial growth factor) content in the solution was detected by ELISA to be 17.6 ng / mL and the Ang-1 (angiopoietin 1) content was 9.3 ng / mL.

[0053] (3) Cut the decellularized bovine Achilles tendon slices into pieces smaller than 1 cm. 2 Small pieces of the decellularized matrix were freeze-dried at -40℃ for 40 h; the small pieces of decellularized matrix were immersed in liquid nitrogen, ground for 2 h, and then heated to 4℃ until the liquid nitrogen completely evaporated to obtain decellularized matrix microparticle powder.

[0054] (4) Add the components according to Table 3:

[0055] Table 3 Component Addition Amount

[0056]

[0057] Weigh the above components according to Table 3, mix them using a homogenizer, and then fill them into syringes to obtain the finished filling and repair agent.

[0058] Comparative Example 1

[0059] The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 does not contain vascular endothelial cell supernatant and is replaced by physiological saline.

[0060] Angiogenesis-promoting function test:

[0061] Take 1g of the finished products from Example 1 and Comparative Example 1 respectively, add 10mL of vascular endothelial cell culture medium, extract at 37℃ for 48h, centrifuge and collect the supernatant to obtain extracts of the two filling repair agents. Add the extracts to 96-well plates coated with vascular endothelial cells, and culture for 48h. Detect the cell proliferation rate using CCK8. The results are shown in Table 4.

[0062] Table 4 Component Addition Amount

[0063]

[0064] Experiments have shown that the extract of Example 1 significantly promotes the proliferation of vascular endothelial cells, while the extract of Comparative Example 1 has no significant effect on the proliferation of vascular endothelial cells. This demonstrates that adding vascular endothelial cell supernatant to the filler repair agent formulation promotes angiogenesis.

[0065] Comparative Example 2

[0066] The difference between Comparative Example 2 and Example 1 is that Comparative Example 2 does not contain decellularized matrix and is replaced by collagen gel at a concentration of 20 mg / mL.

[0067] Collagenase degradation experiment:

[0068] Take 1g of the finished product from Example 1 and Comparative Example 2, and prepare 3 portions of each. Add a solution containing 100 U / mL collagenase to each portion, gently mix by pipetting, and incubate at 37°C for 2 h, 8 h, and 24 h. At each time point, take one portion, centrifuge at 5000g for 10 minutes, collect the precipitate, transfer it to a crucible, dry at 80°C for 6 h, and weigh it. The experimental results are shown in Table 5. Figure 3 As shown.

[0069] Table 5 Weighing Data Records

[0070]

[0071] Experimental results show that Example 1 degrades more slowly than Comparative Example 2, and the filler repair agent containing decellularized matrix microparticles has a better filling and maintenance effect than collagen gel.

[0072] Comparative Example 3

[0073] The difference between Comparative Example 3 and Example 1 is that Comparative Example 3 does not contain sodium hyaluronate or hydroxypropyl methylcellulose, and is replaced by physiological saline.

[0074] Pushing force test:

[0075] Examples 1 and 3 were taken respectively, connected to 23G injection needles, and mounted on the push-pull tester bracket. The syringes were pushed downwards at a speed of 30 mm / min, and the change in pushing force was detected, and curve data were collected. The experimental results are as follows: Figure 4 As shown, A is the extrusion force curve of Example 1, which shows a smooth extrusion process and a relatively stable extrusion force curve, with a maximum extrusion force of 20.084 N. B is the extrusion force curve of Comparative Example 3. Since it does not contain a suspending agent, the thinner liquid is extruded first during the extrusion process, and then gradually becomes thicker, with the extrusion force increasing accordingly. The extrusion force curve does not form a clear inflection point when the liquid is completely extruded, making it impossible to accurately determine the point at which the liquid is completely extruded based on the change in extrusion force. The maximum extrusion force is approximately 80 N. Therefore, it can be seen that the addition of a suspending agent in the formulation of the filler repair agent of the present invention helps the liquid to be smoothly extruded, so as to facilitate the injection of a uniform gel paste into the body.

[0076] Comparative Example 4

[0077] The difference between Comparative Example 4 and Example 1 is that Comparative Example 4 does not contain glycerol and is replaced by physiological saline.

[0078] Post-push observation:

[0079] Take samples from Example 1 and Comparative Example 4, respectively. After sterilization by cobalt-60 irradiation, push the samples onto the table and observe the gel state. Then, gently roll the samples with your fingers to feel the gel state. Figure 5 As shown, A represents the form of the filler repair agent in Example 1. The filler repair agent of Example 1 is smoothly injected, with a fine and uniform gel texture. It can be smoothly dispensed using a syringe fitted with a 23G injection needle. B represents the form of the filler repair agent in Comparative Example 4. This filler repair agent is difficult to inject, has a noticeable granular texture, and liquid precipitates from the gel. It cannot be smoothly dispensed using a syringe fitted with a 23G injection needle, resulting in needle blockage. Therefore, the addition of a protective agent in the formulation of the filler repair agent of this invention helps to form a uniform and fine gel paste. Even after irradiation sterilization, the components remain evenly distributed and have a fine texture, allowing for smooth dispensing through the injection needle during injection.

[0080] This article uses specific examples to illustrate the inventive concept in detail. The description of the above embodiments is only for the purpose of helping to understand the core idea of ​​the present invention. It should be noted that any obvious modifications, equivalent substitutions or other improvements made by those skilled in the art without departing from the inventive concept should be included within the protection scope of the present invention.

Claims

1. A method for preparing a filler repair agent that promotes angiogenesis, characterized in that, Includes the following steps: S1: Passage the vascular endothelial cells cultured to generation P2-P5 onto a matrix gel and culture them statically at 37°C, 5% CO2 concentration, and 5-10% O2 concentration to induce the vascular endothelial cells to form tubes. Observe under a microscope. When more than 80% of the cells have formed tubular branching structures, collect the cell culture medium. S2: After centrifuging the cell culture medium collected in S1 at ultra-high speed, collect the supernatant and concentrate the supernatant; S3: Cut the decellularized matrix into small pieces, freeze-dry them, grind them in liquid nitrogen, and collect the decellularized matrix particles after the liquid nitrogen has completely evaporated. S4: Mix the supernatant after S2 concentration with the decellularized matrix microparticles collected in S3, add suspending agent and protectant, and homogenize to obtain an angiogenesis-promoting filling and repair agent.

2. The method for preparing a pro-angiogenic filler repair agent according to claim 1, characterized in that, In S1, the vascular endothelial cells are one type of human umbilical vein endothelial cells, hematopoietic endothelial cells, vascular endothelial progenitor cells, or human iPS cells induced to differentiate into vascular endothelial cells; the matrix gel is a soluble basement membrane extract of EHS mouse sarcoma, with a matrix concentration of not less than 8 mg / mL; the specific method for matrix gel plating is as follows: the matrix gel is thawed at 4°C overnight, 0.5 mL of matrix gel is placed in a culture flask pre-cooled at 4°C, and after standing at 4°C for 30 min, it is transferred to 37°C and allowed to stand horizontally for 0.5-2 h until the matrix gel is completely solidified, providing a three-dimensional culture environment for the formation of vascular endothelial cells; the culture medium used for culturing vascular endothelial cells is ECM-specific medium, with fetal bovine serum added at a concentration of 0.01-0.5%.

3. The method for preparing a pro-angiogenic filler repair agent according to claim 2, characterized in that, In S1, the specific method for inducing vascular endothelial cells to form tubes is as follows: vascular endothelial cells are resuspended in culture medium, counted, and then prepared to a concentration of 0.5-2 × 10⁻⁶ cells / mL. 5 Add cell suspension to a matrix gel at a density of 5000-20000 cells / cm³. 2 After gently tapping and shaking to mix, place in a low-oxygen environment for static culture for 2-6 hours, observing every hour during this period. Under a microscope, observe that more than 80% of the cells have elongated into tubular branches and the cells are connected to form a network structure.

4. The method for preparing a pro-angiogenic filler repair agent according to claim 3, characterized in that, In step S2, the specific method for collecting the supernatant is as follows: The vascular endothelial cell culture supernatant is aspirated into a centrifuge tube and centrifuged at 100,000-500,000g at 4°C for 1-3 hours to collect the supernatant. The specific method for concentrating the supernatant is as follows: The supernatant is transferred into a concentration tube containing a 30kDa filter membrane and centrifuged at 3000g at 4°C for 10 minutes. After mixing by pipetting, the mixture is centrifuged again. This process is repeated until the concentration is stopped when the VEGF content in the solution is higher than 5 ng / mL and the Ang-1 content is higher than 0.5 ng / mL, as detected by ELISA.

5. The method for preparing a pro-angiogenic filler repair agent according to claim 4, characterized in that, In S3, the decellularized matrix is ​​prepared from allogeneic or xenogeneic tissues, including human, bovine, ovine, swine, or equine tissues. The tissues include dermis, bladder, submucosa of small intestine, tendon, placenta, amnion, or umbilical cord. The specific method for decellularizing the tissue matrix is ​​as follows: after washing the fresh tissue, hair and fat layer are removed. The tissue is then mixed at 37°C with 0.25-1% trypsin solution, 70% isopropanol containing 0.1-0.5% SDS, 70% isopropanol containing 1-2% Triton X-100, and isopropanol. Each time the solution is changed, the tissue is ultrasonically washed 5 times with purified water to remove residual reagents.

6. The method for preparing a pro-angiogenic filler repair agent according to claim 5, characterized in that, In step S3, the specific method for freeze-drying is as follows: the decellularized matrix is ​​cut into pieces smaller than 1 cm. 2 Small pieces of the decellularized matrix were spread flat in a freeze-drying tray and frozen at -40℃ for 6-24 h. Then, they were transferred to a freeze dryer with the temperature set to -40℃ and the vacuum set to 10-100 Pa for 12-48 h. The specific method for grinding in liquid nitrogen was as follows: the freeze-dried decellularized matrix pieces were placed in a mortar and pestle, and liquid nitrogen was poured in until the matrix was completely submerged. The matrix was gently ground in a circular motion with a pestle for 0.5-2 h. During this period, liquid nitrogen was added to ensure that the sample was submerged. After the temperature was raised to 4℃, the liquid nitrogen was allowed to evaporate completely, and the decellularized matrix particles were collected.

7. The method for preparing a pro-angiogenic filler repair agent according to claim 6, characterized in that, In step S4, the suspending agent is one or two of the following: sodium hyaluronate, methylcellulose, carboxymethyl chitosan, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, carbomer, polyvinyl alcohol, poloxamer, gelatin, collagen, and silk fibroin. The concentration of the suspending agent is preferably such that the viscosity of the suspending agent solution is between 300-1000 mPa·s. The protective agent is one or two of the following: sucrose, trehalose, mannitol, glycerol, glycine, arginine, and polyethylene glycol.

8. The method for preparing a pro-angiogenic filler repair agent according to claim 7, characterized in that, In step S4, the concentrated vascular endothelial cell supernatant and decellularized matrix microparticles are mixed at a ratio of (10-30):1 (v / w). After adding suspending agents and protective agents, the mixture is homogenized at 4°C for 0.5-2 h. The mixed paste is collected, poured into a container, and packaged into a pre-filled syringe using a filling machine. After irradiation sterilization, it is used for injection.

9. A filler for promoting angiogenesis prepared by the method of any one of the angiogenesis-promoting filler for repair according to claims 1-8.

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