A soft tissue filler with pro-angiogenic function and its preparation method and application

Abstract

The application provides a preparation method of a soft tissue filler with a function of promoting vascularization, relates to the field of medical biomaterials, and comprises the following steps: taking an abdominal aorta blood vessel, performing alkali treatment, rinsing, decellularization, sterilization, secondary rinsing, shearing, grinding, screening, and compounding with cross-linked sodium hyaluronate gel to obtain a soft tissue filling gel. The soft tissue filler prepared by the method contains abundant elastin and factors for promoting vascularization, and can improve the defects of existing materials in tissue filling and repair.

Description

Technical Field

[0001] This invention relates to the field of medical biomaterials, and more specifically, to a soft tissue filler with angiotensin-promoting function, its preparation method, and its application. Background Technology

[0002] Hyaluronic acid, also known as hyaluronic acid or glucuronic acid, is a naturally occurring macromolecular glycosaminoglycan and a key component for skin hydration. It boasts excellent biocompatibility and safety, holding a significant position in the medical aesthetics field. Injecting hyaluronic acid into the dermis or subcutaneous tissue can quickly fill facial depressions or wrinkles, achieving an immediate filling effect. Unmodified natural hyaluronic acid molecules are soft and easily broken, and are rapidly degraded and metabolized in the body. Cross-linking technology is used to structurally modify its structure, making the hyaluronic acid molecules more stable, slowing down the degradation rate in the body, and prolonging the filling effect.

[0003] As consumers increasingly prioritize the naturalness of cosmetic results, traditional fillers like hyaluronic acid, while providing quick filling, offer only temporary relief and require regular injections to maintain their effects. Regenerative fillers, on the other hand, stimulate the body's own collagen production to achieve a filling effect, resulting in a natural improvement in the skin from the inside out, better aligning with consumers' pursuit of natural beauty. Regenerative materials primarily include poly-L-lactic acid (PLLA), calcium hydroxyapatite (CaHA), polycaprolactone (PCL), polymethyl methacrylate (PMMA), and polyvinyl alcohol (PVA). These materials are chemically synthesized or processed, offering relatively long-lasting soft tissue filling effects and high stability, but they are prone to triggering inflammatory reactions and nodule formation, leading to long-term complications.

[0004] Decellularized matrix materials (ECMs) have shown great potential in the field of tissue fillers due to their natural origin, excellent biocompatibility, and tissue regeneration capabilities. After being decellularized to remove immunogenic components, ECMs retain their natural spatial scaffold, enabling them to guide the host's own cells to migrate, proliferate, and differentiate in a targeted manner, and to synthesize new, functional progenitor tissues. However, their water-soluble state provides insufficient support, and they are easily absorbed by the body after injection, resulting in a significant reduction in filling effect.

[0005] Current tissue fillers primarily focus on inducing collagen regeneration, but the role of blood vessels in tissue repair is also undeniable. Vascularization is a crucial step in tissue regeneration; good vascularization provides the filled area with sufficient oxygen and nutrients, promoting cell proliferation and differentiation, thereby accelerating tissue repair and regeneration. Furthermore, by promoting angiogenesis, fillers can better integrate with host tissues, reducing the loss of effectiveness due to material degradation or absorption. Therefore, there is an urgent need for a soft tissue filler that can both promote blood vessel ingrowth at the filling site, delay in vivo degradation, and stimulate collagen regeneration. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a soft tissue filler with an antivascular function. The soft tissue filler prepared by this method contains abundant elastin and antivascular factors, which can improve the shortcomings of existing materials in tissue filling and repair.

[0007] Another object of the present invention is to provide a soft tissue filler with angiotensin-promoting function, the filler containing abundant elastin and angiotensin-promoting factors.

[0008] The third objective of this invention is to provide an application of a soft tissue filler that promotes collagen production and blood vessel ingrowth, reorganizes newly formed collagen, strengthens the reticular structure of the dermis, and effectively improves skin texture, roughness, and elasticity.

[0009] The technical problem solved by this invention is achieved by the following technical solution.

[0010] On one hand, embodiments of the present invention provide a method for preparing a soft tissue filler with pro-angiogenic function, comprising the following steps: S1. Material collection: Take the abdominal aorta of a mammal and trim the flocculent outer membrane on the blood vessel.

[0011] S2. Alkali treatment: Add alkali solution at a mass-volume ratio of 5-15% and let it stand at 2-8℃ for 10-30 minutes.

[0012] S3. Rinse: Add rinsing solution I at a mass-volume ratio of 5-20% and rinse 5-10 times with shaking at 50-100 r / min to remove fat and impurities around blood vessels. S4. Decellularization: Add decellularization solution I at a mass-to-volume ratio of 5-15%, place in a constant temperature shaker at 25-37℃ and shake at a speed of 100-150 r / min for 10-30 hours. Replace with decellularization solution II at a mass-to-volume ratio of 5-10%, place in a constant temperature shaker at 25-37℃ and shake at a speed of 100-150 r / min for 1-2 hours. S5. Disinfection: Add disinfectant at a mass-volume ratio of 5-15%, place in a constant temperature shaker at 25-37℃, and shake for 1-2 hours at a speed of 50-100 r / min. S6. Secondary rinsing: Add rinsing solution II at a mass-volume ratio of 5-20%, place in a constant temperature shaker at 4-25℃, rotate at 50-100r / min, and rinse 5-20 times to remove decellularization solution I, decellularization solution II, and disinfectant residues. S7. Cutting: Cut the rinsed blood vessels into small pieces; S8. Grinding: Place in a cryogenic grinder and grind at low temperature to obtain decellularized vascular matrix microparticles.

[0013] S9. Sieving: The ground blood vessels are graded and sieved to obtain decellularized vascular matrix microparticles of different particle sizes.

[0014] S10, Composite with cross-linked sodium hyaluronate gel: The prepared decellularized vascular matrix microparticles are added to the cross-linked sodium hyaluronate gel and stirred until homogeneous to obtain a soft tissue filling gel.

[0015] Preferably, in step S1, a fresh animal abdominal aortic blood vessel is taken, placed in a sterile tissue bag, and transported back at a low temperature of 4°C.

[0016] Preferably, in step S2, the alkaline solution is sodium hydroxide with a concentration of 1-5 mol / L.

[0017] Preferably, the rinsing solution I in S3 and the rinsing solution II in S6 are both any one of pure water, physiological saline, phosphate buffer, citrate buffer and carbonate buffer.

[0018] Preferably, in step S4, decellularization solution I is a mixture of sodium chloride, ethylenediaminetetraacetic acid, and 3-[(3-cholamidopropyl)dimethylammonium]propanesulfonic acid, with the pH adjusted to 7.0-7.5 using an alkaline regulator; decellularization solution II is 0.05-1% nuclease. The alkaline regulator is sodium hydroxide solution.

[0019] Preferably, in step S5, the disinfectant is composed of peracetic acid and ethanol. The mass concentration of peracetic acid is 0.05%-2.5%, and the mass concentration of ethanol is 1%-15%.

[0020] Preferably, in step S7, the blood vessel is cut into small pieces of 3mm-5mm.

[0021] Preferably, in step S8, the grinding temperature is -50°C to -20°C, the grinding frequency is 30-60Hz, and the grinding time is 30-60 seconds.

[0022] Preferably, in step S9, the aperture of the sieve is 100-300 μm.

[0023] Preferably, in step S10, the concentration of decellularized extravascular matrix is ​​50-100 mg / mL, and the concentration of sodium hyaluronate in the cross-linked sodium hyaluronate gel is 10-30 mg / mL. The cross-linked sodium hyaluronate gel is a medium-molecular-size injectable modified sodium hyaluronate gel from Aimeike Technology Development Co., Ltd.

[0024] Secondly, embodiments of the present invention provide a soft tissue filler with a pro-angiogenic function, and its application in tissue filling materials.

[0025] Compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects: The soft tissue filler of this invention possesses both the mechanical properties of polymer materials and the functionality of biomaterials. It exhibits excellent biocompatibility and sufficient mechanical support, resulting in a longer-lasting filling effect. It is less prone to diffusion and displacement after injection, allowing for more precise shaping and reducing the likelihood of edema. It promotes collagen production and angiogenesis, with newly generated collagen remodeling and strengthening the dermal reticular structure, effectively improving skin texture, roughness, and elasticity. The newly formed blood vessels provide ample nutrients to fibroblasts, enabling better integration of the filler with the host tissue, mobilizing the body's own repair capabilities, and achieving tissue remodeling.

[0026] The soft tissue filler prepared by this invention is produced using a sterile process, which preserves the natural structure and bioactivity of the decellularized vascular matrix. Furthermore, the method and raw materials provided by this invention are widely available, the preparation process is mature, and large-scale production can be achieved. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a scanning electron microscope (SEM) image of the decellularized vascular matrix. Figure 2 HE staining images of tissue sections of vascular raw materials before and after decellularization; Figure 3 A comparison chart of DNA content before and after decellularization of vascular raw materials; Figure 4 This is a cell morphology diagram of decellularized vascular matrix co-incubated with 3T3 cells; Figure 5 The graph shows the cell survival rate results after co-incubation of decellularized vascular matrix with 3T3 cells; Figure 6 DAPI staining image of decellularized vascular matrix and mesenchymal stem cells co-cultured; Figure 7 HE staining images of graft tissue sections from each group; Figure 8 Masson staining images of tissue sections from each group of grafts; Figure 9 CD31 staining images of graft tissue sections from each group; Figure 10 Sirius red staining images of tissue sections from each group of grafts. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0030] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to specific embodiments.

[0031] Example 1 A method for preparing a soft tissue filler with pro-angiogenic function includes the following steps: (1) Pretreatment: Take the abdominal aorta of a mammal and trim the flocculent outer membrane on the blood vessel.

[0032] (2) Alkali treatment: Weigh the blood vessel fragments and add alkali solution (physiological saline adjusted to pH=10 with 1M sodium hydroxide solution) at a mass-to-volume ratio of 10%, and let stand at 4°C for 30 minutes. Here, the mass-to-volume ratio refers to the ratio of the mass of the blood vessel fragments to the volume of the sodium hydroxide solution. In the embodiments of this invention, the mass-to-volume ratios involved are all the ratio of the mass of the blood vessel fragments to the volume of the added liquid.

[0033] (3) Rinsing: Add rinsing solution I (pure water) at a mass-volume ratio of 15%, place it on a decolorizing shaker in a refrigerator, and rinse 10 times at 100 r / min to remove fat and impurities around the blood vessels.

[0034] (4) Decellularization: Add decellularization solution I (0.5% CHAPS + 0.7% EDTA + 6% NaCl, adjust pH=7.2) at a mass-volume ratio of 10%, place in a constant temperature shaker at 37℃ and shake at 120 r / min for 24 h; replace decellularization solution II (0.05% nuclease) at a mass-volume ratio of 5%, place in a constant temperature shaker at 37℃ and shake at 120 r / min for 2 h.

[0035] (5) Disinfection: Add disinfectant (0.5% peracetic acid + 8% ethanol) at a mass-volume ratio of 15%, place in a constant temperature shaker at 25℃, and shake for 2 hours at a speed of 100r / min.

[0036] (6) Second rinse: Replace with 10% (w / v) rinse solution II (phosphate buffer), and rinse 10 times at 100 r / min to remove cell removal solution I, cell removal solution II and disinfectant residue.

[0037] (7) Cutting: Cut the rinsed blood vessels into small pieces of 3mm-5mm.

[0038] (8) Grinding: The small blood vessel fragments were placed in a cryogenic grinder and ground at low temperature to obtain decellularized vascular matrix microparticles. The grinding temperature was -50℃, the grinding frequency was 60Hz, and the grinding time was 60s. (9) Sieving: Select experimental sieves with different pore sizes, grade and sieve the ground blood vessels, and collect decellularized vascular matrix particles of 100-300μm.

[0039] (10) Combining with cross-linked sodium hyaluronate gel: Decellularized vascular matrix microparticles (100 mg / mL) and cross-linked sodium hyaluronate gel (23 mg / mL) are mixed in a ratio of 3:7 to obtain soft tissue filling gel.

[0040] Experimental Example 1: Detection of relevant indicators of decellularized vascular matrix.

[0041] 1.1 Microstructure of decellularized vascular matrix Cross-sections of decellularized vascular matrix particles were observed using field emission scanning electron microscopy. Samples were first frozen in liquid nitrogen, then freeze-dried, and the sample surface was gold-plated before observation using scanning electron microscopy. The results are as follows: Figure 1 As shown.

[0042] Figure 1 The results showed that the prepared decellularized vascular matrix exhibited an irregular three-dimensional porous structure, which could serve as a scaffold for cell growth, providing channels and space for cell proliferation and internal migration.

[0043] Experimental Example 2: Detection of decellularized vascular matrix tissue sections 2.1 Tissue embedding (1) Take decellularized blood vessels and untreated blood vessel tissue from Example 1 and fix them overnight with 4% paraformaldehyde.

[0044] (2) Remove the tissue from the fixative and dehydrate it in a gradient of alcohols: 75% ethanol for 2 h, 85% ethanol for 2 h, 95% ethanol for 1 h, 95% ethanol for 1 h, 100% ethanol for 30 min, and 100% ethanol for 30 min.

[0045] (3) Transparency: The tissue was placed in xylene for transparency treatment. Xylene I was soaked for 20 min, and xylene II was soaked for 20 min.

[0046] (4) Wax impregnation: The transparent tissue block is placed in a wax tank at 56-58℃ for wax impregnation. Wax tank I is soaked for 1 hour, and wax tank II is soaked for 1 hour.

[0047] (5) Paraffin embedding: First, put the melted wax into the embedding frame. Before solidification, take the tissue out of the dehydration box and put it into the embedding frame and mark it. Cool at -20℃. After solidification, take the wax block out of the embedding frame and trim it.

[0048] (6) Paraffin sections: cut into 5μm thin sections, flatten the tissue in 37℃ warm water, pick up the tissue with a glass slide, bake the sections in a 60℃ oven, and store at room temperature.

[0049] 2.2 HE staining (1) Dewaxing: Soak the tissue sections in xylene for 10 minutes, then replace the xylene and soak for another 10 minutes. Soak in 100%, 95%, 90%, 80%, and 70% alcohol for 5 minutes each, then hydrate. (2) Staining - Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 5-10 minutes, rinse the sections with running water to remove excess staining solution.

[0050] (3) Differentiation: Place the slices in hydrochloric acid alcohol differentiation solution for a few seconds until the slices turn light red.

[0051] (4) Blueing: Rinse immediately with running water, then put into ammonia water for several minutes until the slices turn blue.

[0052] (5) Eosin staining: Immerse the slide in eosin staining solution for 1-2 minutes.

[0053] (6) Dehydration and clearing: Soak in 70%, 80%, 90%, 95%, and 100% alcohol for 5 minutes each to dehydrate. Soak in xylene for 10 minutes, then replace with xylene and soak for another 10 minutes to make the slices clear.

[0054] (7) Sealing: Use neutral resin to seal the film to avoid air bubbles.

[0055] Figure 2 HE staining images of tissue sections of vascular raw materials before and after decellularization. Figure 2 It can be concluded that after decellularization treatment, the cells and nucleic acid substances in the extracellular matrix of vascular tissue are significantly removed, while a large number of cellular components and nucleic acid substances can be seen in vascular tissue that has not undergone decellularization treatment. This shows that the decellularization method in this invention can effectively remove cellular components from vascular tissue and achieve the effect of decellularization.

[0056] Experimental Example 3: Quantitative Detection of DNA 3.1 Extraction and purification of DNA from tissues (1) Take about 10 mg of blood vessels that have been decellularized and rinsed in Example 1 above and untreated blood vessel tissue, freeze-dry them and grind them into powder.

[0057] (2) Add 100 μg / mL proteinase K and digest at 56℃ for 2 h until the sample is completely transparent. Centrifuge at 10000g and 4℃ for 10 min and collect the supernatant.

[0058] (3) Add an equal volume of phenol-chloroform-isoamyl alcohol and gently invert to mix. Centrifuge at 10000g for 10 min at 4℃, and carefully transfer the upper aqueous phase to a new centrifuge tube.

[0059] (4) Add one-tenth of the supernatant volume of 3mol / L sodium acetate and 2.5 times the volume of anhydrous ethanol, and let stand overnight at 4°C to precipitate DNA.

[0060] (5) Centrifuge at 10000g for 10 min, discard the supernatant, evaporate the liquid to obtain the DNA sample.

[0061] 3.2 Quantitative DNA Detection Preparation of DNA standard curve samples: Prepare a 2 μg / mL solution of DNA standard using 1×TE buffer. Measure the absorbance of DNA at 260 nm in a cuvette with a 1 cm optical path. Prepare 400 μL each of the following standard solutions using 1×TE buffer: 0 ng / mL, 1.25 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng / mL. Add 125 μL of the diluted standard solution to each well of a 96-well black ELISA plate, with three replicates per sample.

[0062] Preparation of test samples: Take the purified spiked recovery test DNA purified sample and the test sample DNA purified sample, add 1×TE buffer for appropriate dilution to obtain 400μL of dilution solution, take 125μL of each well and add it to the 96-well black microplate, and set 3 replicates for each sample.

[0063] Add 125 μL of fluorescent dye (mixed with an equal volume of the sample) to each of the above samples, vortex to mix, and react at room temperature in the dark for 5 min. Measure using a fluorescence microplate reader. Measurement conditions: excitation wavelength 480 nm, emission wavelength 520 nm. Plot the obtained data, analyze the graphs, and derive the regression equation. Use 1XTE buffer as the background fluorescence value, and measure and record the fluorescence value of each well.

[0064] Figure 3 This is a comparison chart of DNA content before and after decellularization of vascular raw materials. Figure 3 The results showed that the DNA content in the untreated vascular material was as high as 231.5 ng / mg. After the vascular material was treated with the decellularization method of the present invention, the DNA residue was only 25.3 ng / mg, and the DNA content was significantly reduced, which can effectively reduce immunogenicity.

[0065] Experimental Example 4: In vitro cytotoxicity test 4.1 Preparation of extract: The sample prepared in Example 1 was added to the culture medium at a ratio of 0.1 g / mL and extracted at 37°C for 72 h to obtain the extract.

[0066] 4.2 In vitro cytotoxicity assay: Take normally cultured 3T3 cells and adjust the cell density to 1×10⁻⁶. 5 The extract was inoculated at a rate of 100 μL / mL into 96-well plates, with each well containing 100 μL of extract. The plates were incubated at 37°C and 5% CO2 for 24 h. A negative control, positive control, and blank control group were also included, and all were placed in the same containers and under the same conditions as the samples during the extraction process (37°C, 5% CO2, 24 h). After 24 hours of culture, cell growth and morphology were examined in each well. The culture medium was aspirated, and the corresponding treatment medium was added. Cells were incubated at 37°C with 5% CO2 for 24 hours. Cell morphological changes were observed and recorded under a microscope. The culture medium was then removed, and 100 μL (containing 10 μL of CCK8) was added. Cells were incubated at 37°C with 5% CO2 for 1–4 hours. Absorbance was measured at 450 nm. Cell viability was calculated using the following formula: Survival rate (%) = (OD value of experimental group - OD value of blank control group) / (OD value of control group - OD value of blank control group) × 100%.

[0067] Figure 4 This is a cell morphology diagram after co-incubation of decellularized vascular matrix with 3T3 cells. Figure 4 The results showed that in the positive control group, gaps appeared between cells, and the cells shrank, became rounded, and lost their normal adhesion ability. The experimental group, blank group, and negative group had the same morphology, the cells grew well, adhered tightly to the wall, and the cells showed a state of aggregation or continuous growth. This indicates that the decellularized vascular matrix prepared by the present invention is not toxic to cells.

[0068] Figure 5 This is a result of cytotoxicity in the decellularized vascular matrix. For example... Figure 5 The results showed that the cell survival rate was higher than 90% in the extracts of decellularized vascular matrix at four different concentrations of 25%, 50%, 75%, and 100%. Moreover, the cell survival rate increased significantly with the increase of concentration, which indicates that the decellularized vascular matrix prepared in this invention can promote cell proliferation and differentiation.

[0069] Experimental Example 5: Decellularized Vascular Matrix for Cell Culture To investigate whether decellularized vascular matrix can be used for three-dimensional cell culture, normally cultured mesenchymal stem cells were taken and the cell density was adjusted to 1×10⁻⁶. 5 Cells / mL were added to dried decellularized vascularized matrix in 12-well plates, followed by dropwise addition of cell suspension to the matrix. The blank control group received no treatment. During extraction, the plates were placed in the same containers and under the same conditions as the sample plates (37°C, 5% CO2, 24h). The culture medium in the culture dishes was aspirated, and the cells were gently rinsed twice with pre-chilled DPBS. The cells were fixed with 4% paraformaldehyde, and DAPI working solution was added, ensuring complete coverage of the sample. The plates were incubated at room temperature in the dark for 10 min. The DAPI staining solution was aspirated, and the plates were quickly rinsed 1-2 times with DPBS to remove unbound dye. The cells were resuspended in a small amount of PBS or mounting medium, and 20 μL was dropped onto a glass slide, covered with a coverslip, and observed.

[0070] Figure 6 This is a DAPI staining image of decellularized vascular matrix and mesenchymal stem cells co-cultured. Figure 6 The results showed that mesenchymal stem cells in the blank group were evenly distributed in the culture medium, while mesenchymal stem cells could be observed adsorbed in the porous space scaffold of the component with added decellularized vascular matrix, indicating that the decellularized vascular matrix prepared in this invention can be used for three-dimensional cell culture.

[0071] Experimental Example 6: This soft tissue filler was used for tissue filling. 6.1 Preparation and grouping of experimental materials (1) Material preparation: Acellular vascular matrix material (VEM) was prepared according to the above method. It was mixed with physiological saline to obtain a VEM solution with a concentration of 30 μg / mL. Cross-linked sodium hyaluronate (HA) and injectable physiological saline were purchased from the market. Acellular vascular matrix material and cross-linked sodium hyaluronate were mixed in a ratio of 3:7 to obtain soft tissue filler (VHA).

[0072] (2) Grouping: ① Saline injection material group (blank group), ② Cross-linked sodium hyaluronate material group (HA), ③ Decellularized vascular matrix material group (VEM), ④ Decellularized vascular matrix + cross-linked sodium hyaluronate material group (VHA).

[0073] (3) Subcutaneous injection in animals Ten male rats weighing 200-250g were selected and anesthetized with 4% methylphenidate hydrate. The rats were fixed with their backs facing upwards, and four injection points were selected on both sides of the spine, two on each side, symmetrically. These four injection points corresponded to the four groups mentioned above. The fur on the rats' backs was removed to expose the skin, and the backs were disinfected with alcohol. 0.25mL of the above material was drawn into a syringe and injected into the corresponding sites. After injection, a circular bulge was visible on the rat's back. After injection, the experimental rats were placed in standard animal cages for free movement and feeding.

[0074] (4) Graft collection Rats were randomly selected at 2, 4, and 6 weeks after injection. The rats were euthanized, the skin on the back of the rats was cut open, and the grafts of each group were completely removed. The grafts were photographed and the presence of blood vessels on the surface of the grafts was observed. The volume of the grafts was measured with calipers and then placed in centrifuge tubes containing 4% paraformaldehyde.

[0075] Results Analysis: In the second week after injection, the volume of the HA and VHA groups increased, while the volume of the blank and VEM groups decreased sharply. This was because the water in the injections of the blank and VEM groups was absorbed by the body. As time increased, the graft volume of each group decreased, but the HA group and the VHA group with added HA still had a good volume retention rate. This was because the cross-linked sodium hyaluronate formed a more stable three-dimensional network, thus slowing down the degradation rate.

[0076] 6.2 Tissue section examination 6.2.1 Paraffin embedding and sectioning (1) Fixation and sampling: Fresh graft specimens collected 6 weeks after injection are fixed in tissue fixation solution for more than 48 hours and the specimens are trimmed.

[0077] (2) Dehydration and paraffin immersion: The trimmed specimens are placed in a dehydration box and then placed in a series of ethanols for dehydration. After being cleared by xylene, the specimens are immersed in melted paraffin.

[0078] (3) Paraffin embedding: The tissue that has been soaked in wax is embedded, and the wax block is trimmed after the wax solidifies.

[0079] (4) Sectioning: Place the trimmed paraffin block on a pathological microtome and section it to a thickness of 5 μm.

[0080] (5) Baking the slides: Place the slides on a baking machine and bake them at 65°C to dry the moisture. Store the slices at room temperature for later use.

[0081] 6.2.2 Hematoxylin-eosin (HE) staining (1) Dewaxing and dehydration: The slices were placed in xylene for dewaxing, and then immersed in 95% ethanol I, 95% ethanol II, 80% ethanol and 70% ethanol in sequence for gradient dehydration.

[0082] (2) Staining: Place the sections in hematoxylin staining solution and stain for 5 min, then rinse with running water; differentiate with differentiation solution for 10 s, then rinse with running water; add ammonia to promote blue color, then rinse with running water. Place the sections in 85% and 95% graded ethanol solutions for 5 min each to dehydrate, stain with eosin solution for 5 min, then rinse with running water.

[0083] (3) Dehydration and mounting: The sections are placed in ethanol for gradient dehydration and then cleared with xylene. After the sections are naturally dried, they are fixed with neutral resin.

[0084] Figure 7 HE-stained images of graft tissue sections from each group 6 weeks after implantation. Figure 7 As shown, a small number of cells grew into the filling material in the HA group, which was surrounded by a thin layer of tissue fibers. Fibroblasts and newly formed capillaries were observed to grow into the filling material in both the VEM and VHA groups, with the VHA group having slightly more blood vessels than the VEM group.

[0085] 6.2.3 Ponceau S Acid Fuchsin-Aniline Blue (Masson) Staining (1) Dewaxing and dehydration: The slices were placed in xylene for dewaxing, and then immersed in 95% ethanol I, 95% ethanol II, 80% ethanol and 70% ethanol in sequence for gradient dehydration.

[0086] (2) Staining: Add hematoxylin for 3 min, rinse with running water; add differentiation solution for 3 s, rinse with running water; add ammonia to promote blue color for 1 s, rinse with running water; add virgin red for 5 s, add phosphomolybdic acid for 30 s, remove excess virgin red dye solution, and enhance the binding ability of collagen fibers with aniline blue dye. Rinse with running water immediately after adding aniline blue.

[0087] (3) Dehydration and mounting: The sections are placed in ethanol for gradient dehydration and then cleared with xylene. After the sections are naturally dried, they are fixed with neutral resin.

[0088] Figure 8 Masson staining images of tissue sections from each group. (Example:) Figure 8 As shown, the VHA and VEM groups have a large number of newly formed blue collagen fibers growing into the interior of the filler, with an orderly structure, while the HA group only forms a small number of sparse blue collagen fibers.

[0089] 6.2.4 CD31 Immunohistochemical Staining of Grafts (1) Dewaxing and hydration: The sections were immersed in xylene I, xylene II, anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 80% ethanol, 70% ethanol, and distilled water in sequence; (2) Antigen retrieval: Place the slides in a retrieval box containing EDTA retrieval solution and microwave on medium-high for 2 minutes, then turn off and keep warm for 2 minutes. Repeat this cycle 3 times. After cooling naturally for 30 minutes, place the slides in PBS and rinse 3 times for 5 minutes each time. (3) Blocking endogenous peroxidase: drip 3% hydrogen peroxide solution onto the slices, incubate at room temperature for 10 min, then place the slices in PBS and rinse 3 times; (4) Blocking: Circle the target tissue with an immunohistochemical pen, add blocking solution to the circle, incubate at room temperature for 30 min, and then remove the blocking solution with a micropipette; (5) Primary antibody incubation: Add a specific ratio of primary antibody to the slide, place the slide flat in a humidified chamber and incubate overnight at 4°C. The next day, rinse the slide three times in PBS; (6) Secondary antibody incubation: Add a specific ratio of secondary antibody to the slide and incubate at 37°C for 30 min. After 30 min, rinse the slide three times in PBS; (7) DAB staining: Add DAB staining solution to the tissue section, observe and control the staining time under a microscope, generally 5-12 min. After staining is complete, immerse the section in PBS to stop staining. (8) Counterstaining: hematoxylin for 5 min, differentiate with 1% hydrochloric acid alcohol for 3 s, rinse with running water to reverse blue staining; (9) Dehydration and mounting: Immerse the sections in 70% alcohol, 80% alcohol, 95% alcohol I, 95% alcohol II, anhydrous alcohol I, anhydrous alcohol II, xylene I, and xylene II in sequence; (10) Mount the slide with neutral resin and observe it under a microscope.

[0090] Figure 9 Images show the CD31 immunohistochemical results for each group; blood vessels support local cell growth by transporting nutrients and oxygen, which contributes to tissue repair and regeneration; therefore, blood vessel growth is an indispensable part of tissue repair. Figure 9As shown, a small number of blood vessels appeared in the HA group (arrows). Both the VEM and VHA groups significantly promoted the formation of new blood vessels. This phenomenon may be due to the porous structure of both the VEM and VHA groups, which provides a suitable growth scaffold for new blood vessels.

[0091] 6.2.5 Sirius red staining of grafts (1) Dewaxing and hydration: The sections were immersed in xylene I, xylene II, anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 80% ethanol, 70% ethanol, and distilled water in sequence; (2) Staining: Place the slides in a staining jar containing Sirius red staining solution and stain at room temperature for 30 min. Then rinse the slides under running water for 10 s. (3) Dehydration and mounting: The slides were placed in ethanol for gradient dehydration and then cleared with xylene. After the slides were air-dried, they were fixed with neutral resin and observed under a polarizing microscope.

[0092] like Figure 10 As shown, to better study the formation of new collagen in each group, the grafts were stained with Sirius red, and the formation of new collagen was observed under a polarized light microscope. Type III collagen is the main component of new collagen fibers in the filler. In the VHA group, a large number of type III and type I collagen fibers were observed in the filler, forming an orderly, interwoven network structure. In the HA group, most collagen fibers were red, and less type III collagen was generated. The type III collagen in the VEM group was lower than that in the VHA group, but better than that in the HA group. This is because the VEM group did not provide sufficient support in vivo and was absorbed by the body. The results indicate that the VHA group has a good ability to promote tissue regeneration.

[0093] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for preparing a soft tissue filler with pro-angiogenic function, characterized in that, Includes the following steps: S1. Material collection: Take the abdominal aorta of a mammal and trim the flocculent outer membrane on the blood vessel; S2. Alkali treatment: Add alkali solution at a mass-volume ratio of 5%-15% and let it stand at 2-8℃ for 10-30 minutes. S3. Rinse: Add rinsing solution I at a mass-volume ratio of 5%-20%, and rinse 5-10 times with shaking at 50-100 r / min. S4. Decellularization: Add decellularization solution I at a mass-to-volume ratio of 5%-15%, place in a constant temperature shaker at 25-37℃ and shake at a speed of 100-150 r / min for 10-30 h; add decellularization solution II at a mass-to-volume ratio of 5%-10%, place in a constant temperature shaker at 25-37℃ and shake at a speed of 100-150 r / min for 1-2 h; S5. Disinfection: Add disinfectant at a mass-volume ratio of 5%-15%, place in a constant temperature shaker at 25-37℃, and shake for 1-2 hours at a speed of 50-100 r / min. S6. Secondary rinsing: Add rinsing solution II at a mass-volume ratio of 5%-20%, place in a constant temperature shaker at 4-25℃, rotate at 50-100r / min, and rinse 5-20 times to remove decellularization solution I, decellularization solution II and disinfectant. S7. Cutting: Cut the rinsed blood vessels into small pieces; S8. Grinding: Place in a cryogenic grinder and grind at a low temperature; S9. Sieving: The ground blood vessels are sieved to collect decellularized vascular matrix particles; S10, Combined with cross-linked sodium hyaluronate gel: The prepared decellularized vascular matrix microparticles are added to the cross-linked sodium hyaluronate gel and stirred until homogeneous to obtain a soft tissue filling gel.

2. The method for preparing the soft tissue filler with pro-angiogenic function according to claim 1, characterized in that, In S2, the alkaline solution is sodium hydroxide with a concentration of 1-5 mol / L.

3. The method for preparing the soft tissue filler with pro-angiogenic function according to claim 1, characterized in that, The rinsing solution I in S3 and the rinsing solution II in S6 are any one of pure water, physiological saline, phosphate buffer, citrate buffer and carbonate buffer.

4. The method for preparing the soft tissue filler with pro-angiogenic function according to claim 1, characterized in that, In S4, decellularization solution I is a mixture of sodium chloride, ethylenediaminetetraacetic acid and 3-[(3-cholamidopropyl)dimethylammonium]propanesulfonic acid, with a pH of 7.0-7.5; decellularization solution II is a nuclease.

5. The method for preparing the soft tissue filler with pro-angiogenic function according to claim 1, characterized in that, In step S5, the disinfectant includes peracetic acid and ethanol; the mass concentration of peracetic acid is 0.05%-2.5%, and the mass concentration of ethanol is 1%-15%.

6. The method for preparing the soft tissue filler with pro-angiogenic function according to claim 1, characterized in that, In step S7, the blood vessel is cut into small pieces of 3mm-5mm.

7. The method for preparing the soft tissue filler with pro-angiogenic function according to claim 1, characterized in that, In step S8, the grinding temperature is -50℃ to -20℃, the grinding frequency is 30-60Hz, and the grinding time is 30-60S.

8. The method for preparing the soft tissue filler with pro-angiogenic function according to claim 1, characterized in that, In the S10 described therein, the concentration of decellularized vascular matrix is ​​50-100 mg / mL, and the concentration of sodium hyaluronate in the cross-linked sodium hyaluronate gel is 10-30 mg / mL.

9. A soft tissue filler with pro-angiogenic function, characterized in that, It is prepared by the method described in any one of claims 1-8.

10. The application of a soft tissue filler with pro-angiogenic function prepared by the method of any one of claims 1-8 in tissue filling materials.

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