Composition comprising SVF and hyalurous acid and use thereof for tissue repair

Abstract

The invention provides a composition containing SVF and hyaluronic acid and application of the composition to tissue repair. Furthermore, the invention obtains the angiogenesis peptide for promoting angiogenesis through screening and identification, and the polypeptide has better effects of promoting endothelial cell proliferation and angiogenesis. The composition prepared by mixing the angiogenesis peptide MA-R-5 capable of promoting angiogenesis with SVF and fat particles and adding hyaluronic acid has the advantages of better reducing the degradation of a graft and promoting the generation of new vessels around the graft, and has a wide application prospect.

Description

Technical Field

[0001] This application relates to the biological field, and more specifically to compositions comprising SVF and hyaluronic acid and their use in tissue repair. Background Technology

[0002] SVF (Simplified Cell Fiber) is the sum of cellular components extracted from adipose tissue after collagenase digestion. It is a mixed cell population containing adipocytes, vascular endothelial progenitor cells (ADSCs), monocytes, pericytes, fibroblasts, erythrocytes, hematopoietic stem cells, lymphocytes, macrophages, and extracellular matrix. The ASCs it contains can differentiate in vitro into adipocytes, osteoblasts, chondrocytes, nerve cells, muscle cells, pancreatic cells, and hepatocytes. Furthermore, SVF can secrete various bioactive factors, playing a role in immunomodulation, promoting angiogenesis, and facilitating graft implantation. Due to these characteristics, adipose SVF has achieved significant therapeutic effects in the treatment of chronic diseases such as bone and cartilage repair, systemic sclerosis, skin wound healing, and diabetes.

[0003] Currently, the clinical application of adipose-derived fat fraction (SVF) mainly involves the following steps: collection of autologous adipose tissue, SVF isolation and preparation, and local injection or intravenous infusion. Among these, SVF isolation and preparation is a crucial step affecting quality and therapeutic efficacy. There are two main methods for isolating SVF from adipose tissue: enzymatic digestion and mechanical methods. In 1964, Rodbell first used collagenase digestion to digest collagen fibers from adipose tissue and isolate SVF. Since then, collagenase digestion has been considered the gold standard for SVF extraction from adipose tissue. The yield and viability of SVF cells obtained through enzymatic digestion are closely related to the type and concentration of the digestive enzyme, digestion time, fat aspiration site, liposuction method, and the individual fat content of the donor. Mechanical methods utilize physical forces such as centrifugation, vortexing, and manual shaking to disrupt the integrity of the extracellular matrix (ECM) and surrounding structures of adipose tissue to obtain SVF, which includes cell debris, blood cells, and ECM fragments in addition to cells. Commonly used mechanical methods include centrifugation, oscillation, the use of auxiliary materials, and ultrasound. In 2013, the International Federation for Adipose Therapy and Science (IFATS) and the International Society for Cell Therapy (ISCT) proposed classifying cells extracted from adipose tissue without in vitro culture as SVF. Cells extracted from adipose tissue that have been cultured in vitro and can adhere to a culture vessel are called ASC. SVF cells have the phenotype CD31-CD34+CD45-CD235a-, while cultured ASC cells have the phenotype CD31-CD44+CD45-CD73+CD90+CD105+. Alexander suggested further subdividing SVF extracted from adipose tissue into cSVF and tSVF. tSVF is the product obtained by mechanically destroying the aspirated fat, while cSVF is the product obtained by separating cellular components from other contact materials through collagenase digestion. Research shows that SVF obtained through enzymatic digestion is currently the most widely used in clinical practice because it releases more cellular components, yields a greater quantity of stem (progenitor) cells, and is easier to monitor for quality. In recent years, the use of automated equipment to extract SVF has gradually become the mainstream of clinical application. The emergence of automated equipment has facilitated the SVF extraction process, reduced the risk of human contamination, facilitated the clinical use of SVF, and accelerated the progress of SVF clinical application.

[0004] Adipose-derived SVF contains atherosclerotic cells (ASCs) which have the ability to differentiate into osteoblasts and chondrocytes in vitro and can secrete various cytokines. These cytokines can activate aging and damaged osteoblasts and chondrocytes, repairing damaged bone tissue and thus being used in the clinical regenerative treatment of bone diseases. In a clinical trial of severe arthritis with a one-year follow-up, Lapuente et al. found that the treatment group showed significant improvement in clinical symptoms and a significant decrease in pro-inflammatory factors, while anti-inflammatory factors significantly increased. This indicates that adipose-derived SVF can activate cartilage repair, reduce joint inflammation, and alleviate patient pain through cytokine stimulation. In a clinical trial conducted by the Northern Theater Command General Hospital of China using SVF injection to treat knee osteoarthritis, the treatment group showed significant improvement in knee osteoarthritis symptoms. In a study on the effect of adipose-derived SVF injection dosage on the treatment effect of knee osteoarthritis, Tsubosaka et al. found that the high-dose group was superior to the low-dose group in terms of symptoms and pain. Fujita et al. found that double injections for osteoarthritis showed more significant symptom improvement than single injections, indicating that multiple injections are often more effective for patients with severe conditions.

[0005] Early fat grafting surgeries were not ideal, with many grafts being absorbed, liquefied, or necrotic, leading to complications such as infection, calcification, and cysts. However, the immunomodulatory, cytokine-secreting, and angiogenesis-promoting effects of fat SVF can improve graft retention and success rates. Therefore, the combined transplantation of autologous fat and SVF has been widely applied in the field of regenerative plastic surgery, including facial filling, breast reconstruction, and facial and hand rejuvenation. In a clinical trial conducted by Yin et al., involving localized fat grafting to the face, the graft survival rate in the SVF transplantation group was significantly higher than that in the control group, with better improvement in facial skin. Regarding the ratio of fat to SVF transplantation, scientists have found through clinical trials that the optimal ratio of fat particles to SVF is 1:1, resulting in the lowest fat absorption rate. In summary, relying on the angiogenesis and immunomodulatory functions of fat SVF can improve the retention rate of autologous fat grafts, a function that has been widely applied and has shown good therapeutic effects in the field of plastic surgery.

[0006] Procházka et al. isolated factors secreted by ADSCs, prepared concentrated therapeutic factors, and injected them into ischemic limbs of rabbits. They found that the blood perfusion of ischemic tissue in the experimental group was twice that of the control group; immunohistochemistry showed that the capillary density in the experimental group was significantly higher than that in the control group. This indicates that the cytokines secreted by ADSCs can promote angiogenesis. In fact, after fat transplantation, ADSCs can effectively secrete a large number of pro-angiogenic factors and anti-apoptotic factors, such as HGF, bFGF, VEGF, PDGF-B, and TGF-β. Among them, VEGF can activate endothelial progenitor cells; induce endothelial cells to express integrin 1, αv, β3, β5 and their ligands, secrete various cathepsins, and degrade the extracellular matrix; together they promote the proliferation, migration and fusion of new blood vessels of endothelial cells, and inhibit endothelial cell apoptosis. HGF, after binding to its receptor, promotes the proliferation of vascular endothelial cells by activating the Grb2 / Sos-Ras-Raf-MAPK signaling pathway. bFGF can induce VEGF expression through the FGFR1 (fibroblast growth factor receptor 1) / c-Src / p38 / NF-κB (nuclear factor-κB) signaling pathway; simultaneously, activation of nuclear factor-κB can promote endothelial cell DNA synthesis, cell division and proliferation, and angiogenesis. TGF-β contributes to the production of extracellular matrix and promotes the interaction between endothelial cells and parietal cells, which is beneficial to angiogenesis.

[0007] Currently, there have been some studies on using stromal vascular fraction (SVF) to promote angiogenesis and adipocyte differentiation. However, the use of SVF in combination with other substances for use in breast implants to simultaneously promote angiogenesis and facilitate implant activation and treatment is an important research direction. Summary of the Invention

[0008] In one aspect, the present invention provides an angiogenic peptide MA-R-5, which promotes angiogenesis, and its amino acid sequence is shown in SEQ ID NO: 1.

[0009] Furthermore, the angiogenic peptides of the present invention can be applied to breast fillers to promote the colonization and angiogenesis of the fillers.

[0010] Furthermore, the present invention discloses a composition comprising SVF and hyaluronic acid, characterized in that it contains angiogenic peptide MA-R-5, which promotes angiogenesis, and its amino acid sequence is shown in SEQ ID NO: 1.

[0011] Furthermore, the present invention provides a composition for tissue repair, characterized in that it contains angiogenic peptide MA-R-5, which promotes angiogenesis, as well as SVF, hyaluronic acid, and fat particles, wherein the amino acid sequence of MA-R-5 is shown in SEQ ID NO: 1.

[0012] Alternatively, the polypeptide of the present invention comprises or is composed of no more than 9, more preferably no more than 8, more preferably no more than 7, more preferably no more than 6, more preferably no more than 5, more preferably no more than 4, more preferably no more than 3, more preferably no more than 2, more preferably no more than 1 added sequence compared to SEQ ID NO: 1.

[0013] Suitablely, the polypeptide of the present invention comprises or is more suitably composed of a sequence that shares 10%, 15%, 25%, 30%, 40%, 45%, 55%, 60%, 70%, 75%, 85%, 90% or greater sequence identity with SEQ ID NO: 1.

[0014] The polypeptides of the present invention can also be formulated individually as pharmaceutical compositions. The pharmaceutical compositions can be suitably formulated for oral, intramuscular, subcutaneous, or intravenous delivery. The pharmaceutical compositions of the present invention can be in various forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. Solid dosage forms are preferred. The polypeptides, pharmaceutical compositions, or constructs of the present invention can incorporate excipients and be used in the form of ingestible tablets, lozenges, tablets, capsules, elixirs, suspensions, syrups, crystals, etc.

[0015] Typically, pharmaceutical compositions comprise the polypeptide or construct of the present invention and a pharmaceutically acceptable diluent or carrier. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate-buffered saline, dextran, glycerol, ethanol, and combinations thereof. Pharmaceutically acceptable carriers may also contain small amounts of excipients that enhance the shelf life or effectiveness of the polypeptide or construct of the present invention, such as humectants or emulsifiers, preservatives, or buffers. Pharmaceutical compositions may comprise anti-adhesives, binders, coatings, disintegrants, flavoring agents, coloring agents, lubricants, adsorbents, preservatives, sweeteners, lyophilization excipients (including lyophilization protectants), or compression aids.

[0016] The polypeptides, constructs, or pharmaceutical compositions of the present invention can be formulated for injection by dissolving, suspending, or emulsifying them in aqueous or non-aqueous solvents, such as vegetable oils or other similar oils, fatty acid glycerides, esters of higher fatty acids, or propylene glycol; and, if desired, have conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives. Acceptable carriers, excipients, and / or stabilizers are non-toxic to the recipient at the doses and concentrations used and include buffers such as phosphoric acid, citric acid, and other organic acids; antioxidants, including ascorbic acid, glutathione, cysteine, methionine, and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methylparaben or propylparaben, benzalkonium chloride, or combinations thereof); and amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, and phenylalanine. Acids, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents, such as EDTA; sugars, such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbitol, raffinose, glucosamine, N-methylglucosamine, galactosamine and neuraminic acid; and / or nonionic surfactants, such as polysorbate, POE ether, poloxamer, Triton-X or polyethylene glycol.

[0017] The tonicity of a pharmaceutical composition can be altered by including a tonic modifier. Such tonic modifiers can be charged or uncharged chemicals. Typical uncharged tonic modifiers include sugars or sugar alcohols or other polyols, preferably trehalose, sucrose, mannitol, glycerol, 1,2-propanediol, raffinose, sorbitol, or lactitol (especially trehalose, mannitol, glycerol, or 1,2-propanediol). Typical charged tonic modifiers include salts, such as sodium, potassium, or calcium ions combined with chloride, sulfate, carbonate, sulfite, nitrate, lactate, succinate, acetate, or maleate ions (especially sodium chloride or sodium sulfate); or amino acids such as arginine or histidine. Suitably, aqueous formulations are isotonic, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" means a solution having the same tonicity as some other solution it is compared to, such as physiological saline or serum. The tensioning agent can be used in amounts from about 5 mM to about 350 mM, for example, in amounts from 1 mM to 500 nM. Suitably, at least one isotonic agent is included in the composition.

[0018] Furthermore, the present invention provides a filler or composition for breast augmentation, wherein the filler or composition is a mixture of SVF and fat particles, such that the concentration of the SVF is (1-8)×10⁻⁶. 6The product contains 1-20 mg / mL of hyaluronic acid and a final concentration of (10-100) μg / mL of the angiogenic peptide MA-R-5.

[0019] In some embodiments, the tissue filler is introduced into tissue voids or defects under sterile conditions. In some embodiments, the tissue filler can be introduced into tissue voids or defects via injection (e.g., using one or more single-tube syringes, double-tube syringes, and / or combinations thereof). In other embodiments, the tissue filler may be initially applied to an external mold (e.g., in a surgical setting) to form a molded component, which can then be removed from the mold and implanted into a patient. Regenerative medicine approaches, including tunable in-situ formed biomaterials, have the potential to address many of these design considerations. In particular, type I oligomeric collagen (oligomers), a highly purified molecular form of collagen readily soluble in dilute acids, represents a tunable in-situ formed biomaterial with the potential to address many of these design considerations. Unlike conventional monomeric collagen formulations (i.e., telogen and detelogenated collagen), oligomers represent small aggregates of full-length triple-helix collagen molecules (i.e., procollagen) with intact carboxyl- and amino-terminal telopeptides held together by naturally occurring intermolecular crosslinks. Retaining these key molecular features, including carboxyl- and amino-terminal telopeptide regions and associated intermolecular crosslinks, provides this natural polymer and the collagen materials it forms with desirable but uncommon properties. More specifically, the oligomers retain the fibrillation (self-assembly) capability inherent in fibrous collagen. After neutralizing the oligomers to physiological conditions (e.g., pH and ionic strength), this liquid form is readily injectable to fully fill complex contours and geometries. At body temperature, the liquid rapidly transforms into a fibrous collagen matrix, recreating the structural and biosignaling features of the collagen matrix found in the extracellular matrix (ECM) components of tissues. Following implantation in vivo, these matrices persist, exhibiting slow metabolic renewal and remodeling, resistance to proteolytic degradation, and no active biodegradation or foreign body response. This natural polymer supports the creation of materials with a wide range of tunable physical properties, including geometry, architecture (random or arranged fibrils, continuous fibrillary density gradients), and mechanical integrity, making it an enabling platform for personalized regenerative medicine. In-situ formed collagen matrix shows promise as a regenerative tissue filler for breast-conserving surgery and other soft tissue restoration needs.

[0020] In some embodiments, collagen used in the methods and compositions described herein may be lyophilized and then reconstituted to form a collagen composition for mixing with a buffer solution as described herein. In such embodiments, the reconstitution of the lyophilized collagen is not a mixing step for collagen polymerization. The term "lyophilized" as used herein means the removal of water from a protein, compound, or composition by, for example, freeze-drying under vacuum. Any lyophilization method known to those skilled in the art can be used. In some embodiments, collagen may be lyophilized in an acid, such as acetic acid, hydrochloric acid, formic acid, lactic acid, citric acid, sulfuric acid, or phosphoric acid. In other embodiments, collagen may be lyophilized in water. In further embodiments, a cryoprotectant or a lyophilization protectant, or a combination thereof, may be used during lyophilization.

[0021] In some embodiments, the lyophilized fat particles can be reconstituted to form the hyaluronic acid composition described herein for mixing with a buffer solution to obtain hyaluronic acid. In some embodiments, the hyaluronic acid can be reconstituted in an acidic solution or in water. In some embodiments, the acidic solution may comprise acetic acid, hydrochloric acid, formic acid, lactic acid, citric acid, sulfuric acid, or phosphoric acid. In some embodiments, the acidic solution for reconstitution may have an acid concentration of about 0.005N to about 0.1N, about 0.005N to about 0.08N, about 0.005N to about 0.06N, about 0.005N to about 0.04N, about 0.005N to about 0.02N, about 0.005N to about 0.01N, or about 0.01N. In some embodiments, the acid may be hydrochloric acid, and the hydrochloric acid may be about 0.005N to about 0.1N hydrochloric acid. In other embodiments, the acid may be hydrochloric acid, and the hydrochloric acid may be about 0.01N hydrochloric acid.

[0022] Furthermore, the filler provided by this invention can be used for tissue repair, including skin damage, breast augmentation, etc.

[0023] Beneficial effects

[0024] This invention provides a composition comprising SVF and hyaluronic acid and its use in tissue repair. Furthermore, this invention has identified a pro-angiogenic peptide through screening, which exhibits good efficacy in promoting endothelial cell proliferation and angiogenesis. The composition prepared by mixing the pro-angiogenic peptide MA-R-5 with SVF and fat particles, and then adding hyaluronic acid, shows good results in reducing graft degradation and promoting the formation of new blood vessels around the graft, demonstrating broad application prospects. Attached Figure Description

[0025] Figure 1 Figure showing the effect of angiogenic peptide MA-R-5 on the proliferative activity of HUVECs.

[0026] Figure 2The effect of angiogenic peptide MA-R-5 on the length of the inferior intestinal vein (SIV) in zebrafish embryos (Figure 1) Detailed Implementation

[0027] Specific embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art.

[0028] Example 1: Screening and Identification of Angiogenic Peptides

[0029] Fresh eels were washed, crushed, homogenized, heated at 65℃ for 25 min, centrifuged at 12000 r / min for 30 min, and the supernatant was added with 3 volumes of pre-cooled acetone and allowed to stand overnight. After centrifugation, the supernatant was removed by rotary evaporation at 30℃, freeze-dried, and reconstituted with phosphate buffer (PBS, 0.05 mol / L NaH2PO4, 0.15 mol / L Na2HPO4, pH 7.2) to obtain the eel polypeptide extract. The eel polypeptide extract was first subjected to Sephadex G-25 column chromatography. The separated fractions were analyzed by in vitro HUVECs proliferation activity testing, and fraction 3 showed the fastest proliferation-promoting properties. Fragment 3 was further separated using Superdex 75Increase 10 / 300GL column chromatography. Fragment 2, exhibiting the strongest pro-proliferative properties, was further purified by HPLC and then subjected to amino acid sequence identification by Biotech. Database comparison revealed the amino acid sequence of the pro-angiogenic peptide as shown in SEQ ID NO: 1, named MA-R-5. Simultaneously, the peptide was artificially synthesized with a purity of 96.8%, and its concentration was adjusted to 1 mg / mL for later use.

[0030] Example 2: Identification of the activity of the angiogenic peptide MA-R-5, which promotes angiogenesis.

[0031] HUVECs (catalog number 04-001-1ACS, Qianshe Biotechnology) in the logarithmic growth phase were prepared to a concentration of 5×10⁻⁶. 4Cell suspensions of varying concentrations were seeded into 96-well plates at 180 μL per well and incubated at 37°C in a 5% CO2 incubator. After 24 h of culture, 20 μL of different concentrations of angiogenic peptide MA-R-5 (10 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml), positive control (bFGF, 10 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml), and control medium were added. All groups were cultured for another 48 h, and 50 μL of 5% MTT solution was added to each well. The plates were incubated at 37°C for 4 h, the culture medium was aspirated, and 150 μL of dimethyl sulfoxide (DMSO) was added. After gentle shaking for 30 min, the absorbance was measured at 570 nm. The proliferation rate was calculated using the following formula: Proliferation rate = (A-drug group / A-control group) × 100%. A-drug group and A-control group represent the optical density values ​​of the drug-treated group and the control group, respectively. Results are as follows: Figure 1 As shown.

[0032] from Figure 1 It can be seen that the angiogenic peptide MA-R-5 has a strong pro-proliferative effect on HUVEC cells, and the pro-proliferative effect increases with increasing concentration, showing a dose-dependent relationship. Figure 1 The proliferation-promoting activity was highest at a concentration of 100 μg / ml, with a proliferation rate of (145.23±1.04)%, which was better than that of the control group bFGF.

[0033] Example 3: Angiogenic activity experiment of the pro-angiogenic peptide MA-R-5 on angiogenesis.

[0034] Healthy zebrafish embryos were collected and grouped into 24-well plates (20 embryos / well). They were placed in amniotic fluid (filtered pure water) and cultured in an incubator at 27°C. At the 20th hour of embryonic development, the egg membrane was removed by digestion with 1 g / L trypsin at room temperature for 5 min. At the 24th hour of embryonic development, different concentrations of MA-R-5 polypeptide solution (10 μg / ml, 50 μg / ml, 100 μg / ml, and 200 μg / ml) were added. The positive control was bFGF (50 μg / ml), and the negative control was amniotic fluid only. After 12 hours of treatment, the amniotic fluid was replaced. The staining method was as follows: Embryos developed to 72 hpf were fixed with 4% paraformaldehyde for 2 h, washed twice with PBS (pH 7.0), and then sequentially immersed in a mixture of methanol and PBT buffer (0.1% Triton X-100, 0.05% BSA in PBS solution) for osmotic dehydration (methanol mass fraction gradient of 25%, 50%, 75%, 100%), followed by immersion in the opposite gradient for 15 min each time. The embryos were then placed in NTMT buffer to equilibrate before staining. 375 μg NBT and 200 μg X-phosphate were added to each milliliter of NTMT to ensure all zebrafish embryonic blood vessels were labeled. PBST was added to terminate the staining reaction. The embryos were then immersed in a PBS solution containing 5% formaldehyde amine and 10% hydrogen peroxide for 30 min to remove endogenous melanin from pigment cells and make the stained blood vessels visible. Blood cell flow in the embryonic blood vessels was observed under a stereomicroscope, images were acquired, and the length of the inferior venous vein (SIV) was calculated. Results are as follows: Figure 2 As shown.

[0035] from Figure 2 It can be seen that the angiogenic peptide MA-R-5, which promotes angiogenesis, gradually enhances its growth-promoting effect on the inferior venous vein (SIV) of zebrafish embryos with increasing concentration. However, the proliferative effect gradually weakens after exceeding 100 μg / ml. Therefore, 100 μg / ml is the optimal concentration for promoting growth. At a concentration of 100 μg / ml, the length of the zebrafish embryonic SIV reached (13.2 ± 0.41) × 100 μm, while the control at the same concentration only reached (9.9 ± 0.36) μm.

[0036] ×100μm. The above results demonstrate that the angiogenic peptide MA-R-5 has excellent angiogenesis-promoting effects.

[0037] Example 4: Preparation of a composition containing the angiogenic peptide MA-R-5, SVF, and hyaluronic acid.

[0038] Preparation of SVF: Inguinal adipose tissue was aseptically obtained from four 6-8 week old male mice; after washing the adipose tissue with PBS, 1 mL of PBS was added, and the tissue was cut into 1 mm pieces. 3 Size; Add 3 mL of 0.1% collagenase digestion solution to digest adipose tissue particles; shake every 10 minutes, and after 70 minutes, transfer out all tissue digestion mixtures. After digestion, filter through a 200-mesh filter. After filtration, centrifuge the suspension at 1200g for 7 minutes, discard the supernatant, and retain the liquid adhering to the lower end of the centrifuge tube as the SVF cell population. Resuspend in 1 mL of physiological saline, observe cell morphology under an inverted phase-contrast microscope, count cells using a cell counter, and perform trypan blue staining. The cell viability was calculated to be 95%, suitable for subsequent experiments.

[0039] Preparation of fat particles: Inguinal adipose tissue from male mice was homogenized with physiological saline, centrifuged at 1000-2000 r / min at 20℃ for 10 min, and the liquid portion was removed to obtain solid fat particles. Connective tissue was then removed from the obtained fat particles, and large pieces were cut into smaller pieces using tissue scissors, ideally allowing for normal aspiration with a 1 ml syringe after needle removal. The adipose tissue was centrifuged to further improve its purity and was then used for further processing.

[0040] Preparation of the composition for experimental group 1: SVF was mixed with fat particles to achieve a SVF concentration of 2 × 10⁻⁶. 6 The concentration of the product is 10 mg / mL, with added hyaluronic acid at 10 mg / mL and the final concentration of the angiogenic peptide MA-R-5 at 100 μg / mL.

[0041] Preparation of the composition for experimental group 2: An equal amount of fat particles as in experimental group 1, with hyaluronic acid added at a concentration of 10 mg / ml and the angiogenic peptide MA-R-5 at a final concentration of 100 μg / ml.

[0042] Preparation of the positive control group composition: SVF was mixed with fat particles to achieve a SVF concentration of 2 × 10⁻⁶. 6 The concentration of bFGF is 100 μg / mL, with 10 mg / mL of hyaluronic acid added.

[0043] The negative control group consisted of an equal amount of fat particles.

[0044] Example 5: Animal experiments on each group of compositions

[0045] Rats were anesthetized preoperatively with an intraperitoneal injection of 10% chloral hydrate (3 ml / kg), and the surgical area was disinfected. A 1 cm skin incision was made in the midline of the sternum, and the tissue was dissected to the area below the bilateral mammary glands. 0.5 ml of the grafts prepared in Example 4 were placed in the space between the mammary gland and the pectoral muscle. Specifically, in Experimental Group 1: Group 1 grafts were placed in the space behind the right upper mammary gland. In Experimental Group 2: Group 2 grafts were placed in the space behind the right upper mammary gland. In Experimental Group 3: Group 4 grafts were placed in the space behind the right upper mammary gland (positive control group). A total of 5 grafts were performed in each group. After 12 weeks, the rats were anesthetized and dissected. The fat volume preservation rate of each group was calculated, specifically by measuring the specimen volume using the water displacement method; the volume preservation rate = graft volume after 12 weeks / graft volume on the day of transplantation. The results are shown in Table 1.

[0046] Table 1. Volume retention rate of each group

[0047] Group Volume retention rate (%) Experimental group 1 65.83±2.56 Positive control 49.82±1.55 Experimental group 2 60.24±2.13 negative control 21.17±0.73

[0048] Twelve weeks later, rats were dissected and observed to have new fat formation in experimental groups 1 and 2 as well as the positive control group. The results in Table 1 show that when SVF is used together with fat particles, hyaluronic acid and angiogenic peptide MA-R-5, it can effectively improve the volume preservation rate of fat particles, reduce liquefaction, and has a good effect on promoting graft colonization.

[0049] Further, the capsule and adhering connective tissue were separated, the graft was removed, and the microvessel density was counted: the average blood vessel density of 5 fields under 100 high magnification. The microvessel density count results are shown in Table 2.

[0050] Table 2. Microvessel density results for each group

[0051] Group Microvessel density counting Experimental group 1 11.52±0.48 Positive control 8.06±0.27 Experimental group 2 9.15±0.36 negative control 1.34±0.12

[0052] As shown in Table 2, both experimental groups 1 and 2 exhibited good effects in promoting microvascular formation, with higher microvascular density than the positive control. This indicates that the SVF of this invention, together with fat particles, hyaluronic acid, and the angiogenic peptide MA-R-5, can effectively promote fat colonization and microvascular formation, demonstrating promising application prospects.

[0053] Although embodiments of the present invention have been described above in conjunction with the accompanying drawings, the present invention is not limited to the specific embodiments and application fields described above. The specific embodiments described above are merely illustrative and instructive, and not restrictive. Those skilled in the art can make many other forms based on the guidance of this specification and without departing from the scope of protection of the claims of the present invention, and all of these are within the scope of protection of the present invention.

Claims

1. An angiogenic peptide MA-R-5 that promotes angiogenesis, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. Use of the angiogenic peptide MA-R-5 as described in claim 1 in the preparation of a drug that promotes angiogenesis.

3. A filler or composition for breast augmentation, wherein the filler or composition is a mixture of SVF and fat particles, such that the concentration of the SVF is (1-8)×10⁻⁶. 6 The product contains 1-20 mg / mL of hyaluronic acid and a final concentration of (10-100) μg / mL of the angiogenic peptide MA-R-5.

4. Use of angiogenic peptide MA-R-5, which promotes angiogenesis, in the preparation of a composition for tissue repair, wherein the composition further comprises SVF, hyaluronic acid, and fat particles, wherein the concentration of SVF is (1-8)×10⁻⁶. 6 The concentration of the angiogenic peptide MA-R-5 is (10-100) μg / ml, and the dosage of hyaluronic acid is 1-20 mg / ml. The amino acid sequence of MA-R-5 is shown in SEQ ID NO:

1.

5. The use as described in claim 4, characterized in that... The tissue repair referred to is skin damage repair.

6. The use as described in claim 4, characterized in that... The tissue repair referred to is breast repair.

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