A method for preparing a tissue filler based on elastin-like proteins and its use

Abstract

This application belongs to the field of biomaterials technology, specifically disclosing a method for preparing and applying a tissue filler based on elastin-like proteins. The preparation method includes the following steps: dispersing hyaluronic acid in ultrapure water to obtain an aqueous hyaluronic acid solution; dispersing elastin-like proteins in ultrapure water to obtain an aqueous elastin-like protein solution; dispersing glutaraldehyde in ultrapure water to obtain an aqueous glutaraldehyde solution; mixing the aqueous hyaluronic acid solution and the aqueous elastin-like protein solution to obtain a mixture; then adding the aqueous glutaraldehyde solution to the mixture, stirring, and adding composite microspheres to obtain the tissue filler. In this application, the miscibility of hyaluronic acid and elastin-like proteins improves the viscosity of the collagen solution. The resulting gel, after cross-linking with glutaraldehyde, is softer than the gel formed by elastin-like proteins and glutaraldehyde alone. When filled with composite microspheres, it exhibits no foreign body sensation and is stable under acidic conditions.

Description

Technical Field

[0001] This application relates to the field of biomaterials technology, and in particular to a method for preparing and applying an elastin-based tissue filler. Background Technology

[0002] Human skin tissue maintains its structure through an extracellular matrix containing proteins such as collagen or elastin and glycosaminoglycans. When soft tissue is subjected to external impact or aging, fillers can be injected to increase and expand the volume of soft tissue, thereby repairing and correcting deformed soft tissue shapes in a non-surgical manner.

[0003] Existing fillers include substances such as hyaluronic acid and collagen. Hyaluronic acid is composed of polysaccharides similar to those that make up the human body, such as N-acetyl-D-glucosamine and D-glucuronic acid. It is usually a sterile filling product, which requires very strict intermediate process control without terminal sterilization. The production management cost is high, and the guarantee of the sterility of the final product is low.

[0004] Furthermore, hyaluronic acid degrades too quickly, making it difficult to maintain its clinical efficacy after implantation. Even cross-linked sodium hyaluronate gel only maintains its degradation time for about six months. Therefore, there is an urgent need for a tissue filler with a longer degradation time that can be used for medical or cosmetic purposes. Summary of the Invention

[0005] To address the issue of rapid degradation of hyaluronic acid, which makes it difficult to maintain the clinical efficacy after implantation, this application provides a method for preparing and applying a tissue filler based on elastin.

[0006] In a first aspect, this application provides a method for preparing an elastin-based tissue filler, employing the following technical solution:

[0007] A method for preparing an elastin-based tissue filler includes the following steps:

[0008] (1) Disperse hyaluronic acid in ultrapure water and stir until homogeneous to obtain an aqueous solution of hyaluronic acid;

[0009] (2) Disperse the elastin-like protein in ultrapure water and stir until homogeneous to obtain an elastin-like protein aqueous solution;

[0010] (3) Disperse glutaraldehyde in ultrapure water and stir until homogeneous to obtain an aqueous solution of glutaraldehyde;

[0011] (4) Mix the hyaluronic acid aqueous solution from step (1) and the elastin-like aqueous solution from step (2) for 2-3 hours to obtain a mixture. Then add the glutaraldehyde aqueous solution from step (3) to the mixture and continue stirring for 5-20 minutes. Add the composite microspheres and stir for 30-35 minutes to obtain the tissue filler.

[0012] The method for preparing the composite microspheres includes the following steps:

[0013] (1) Disperse chitosan in acetic acid solution and stir at 60-65℃ for 30-50 min. Then add sodium dodecyl sulfate and continue stirring to obtain a mixture.

[0014] (2) Disperse nano-SiO2 in sodium hydroxide solution, stir at 80-85℃ for 1-2 hours, wash with water, then disperse in anhydrous ethanol, add carbon nanotubes, sonicate for 2-3 hours, and dry to obtain a mixture;

[0015] (3) Spray the mixture from step (1) onto the surface of the mixture from step (2) and dry it at 90-95℃ for 2-3 hours to obtain composite microspheres.

[0016] By employing the above technical solution, hyaluronic acid, elastin-like protein, and glutaraldehyde are dispersed in ultrapure water, resulting in aqueous solutions with good dispersibility, uniformity, and stability. Then, the hyaluronic acid and elastin-like protein aqueous solutions are mixed to improve the viscosity of the collagen solution, resulting in an aqueous solution with good stability. Further cross-linking with glutaraldehyde produces a gel that is softer than the gel formed by elastin-like protein and glutaraldehyde alone, exhibiting better mechanical properties. After filling, there is no foreign body sensation, and the gel is stable under acidic conditions, providing a longer effective filling time.

[0017] The composite microspheres loaded in the network structure formed by hyaluronic acid, elastin-like protein and glutaraldehyde increase the elasticity of the gel. The composite microspheres have a certain degree of support, which enhances the support performance of the gel and thus improves the effective filling time of the tissue filler.

[0018] Chitosan has good bioactivity and biocompatibility, and can affect cell metabolism and cell growth, thus playing a role in repairing and moisturizing the skin. Chitosan is soluble in acetic acid and has good water solubility, biocompatibility and film-forming properties. The addition of sodium dodecyl sulfate as a cross-linking agent forms a cross-linked network structure with chitosan, which improves the elasticity of chitosan.

[0019] As a bioactive glass, nano-SiO2, when injected into the skin, can repair, replace, and regenerate body tissues, and can form bonds between tissues and materials. The degradation products of nano-SiO2 can promote the production of skin growth factors, promote cell proliferation, enhance the gene expression of osteoblasts, and promote bone tissue growth. When nano-SiO2 is dispersed in sodium hydroxide solution, the sodium hydroxide solution erodes the nano-SiO2 to a certain extent, increasing the specific surface area and porosity of the nano-SiO2, which is conducive to the loading of carbon nanotubes. The carbon nanotubes loaded on the surface and in the pores of nano-SiO2 improve the mechanical properties of nano-SiO2 and enhance its supporting properties.

[0020] Spraying the mixture onto the surface of the mixture allows the mixture to coat the mixture, increasing the connectivity between nano-SiO2 and carbon nanotubes. This enables the carbon nanotubes to be firmly loaded on the surface of nano-SiO2, thereby improving the mechanical stability of the composite microspheres. This is beneficial for subsequent application in the skin, improving the stability of the composite microspheres in the skin, and thus prolonging the effective filling time of the tissue filler.

[0021] Preferably, the tissue filler includes hyaluronic acid, elastin-like substances, glutaraldehyde, and composite microspheres.

[0022] By employing the above technical solutions, hyaluronic acid possesses strong water-locking properties, which can fill the skin, increase skin volume, and make the skin appear fuller, plumper, and more elastic. Elastin-like proteins are artificially synthesized protein polymers that mimic the amino acid sequence characteristics of natural elastin. They have good biocompatibility and elasticity, and when applied to the skin, they can provide the skin with the ability to resist repeated compression and deformation. Glutaraldehyde, as a collagen coagulant, provides point support after being implanted into the skin, making the skin firmer and having a very good lifting and shaping effect. After implantation, it does not deform or shift, and its support is even stronger.

[0023] Hyaluronic acid and elastin-like proteins are miscible and mix, which improves the viscosity of collagen solutions. Further cross-linking with glutaraldehyde forms a gel that is softer than the gel formed by elastin-like proteins and glutaraldehyde alone. This results in a filling effect without any foreign body sensation, and the gel is stable under acidic conditions, exhibiting good bioactivity. The cross-linking of hyaluronic acid, elastin-like proteins, and glutaraldehyde creates a filler that provides cell nutrition and promotes fibroblast regeneration for a longer period. The filler's effectiveness extends beyond this point, lasting for more than six months. In addition to its basic filling effect, it also retains the benefits of hyaluronic acid. Composite microspheres loaded within the cross-linked network of hyaluronic acid, elastin-like proteins, and glutaraldehyde exhibit good elasticity and support, enhancing the elasticity of the tissue filler. When applied to the skin, they provide better support, thus improving the skin's effectiveness and extending the filling time.

[0024] Preferably, the concentration of the hyaluronic acid aqueous solution is 1-5 mg / mL, the concentration of the elastin-like aqueous solution is 50-650 mg / L, and the concentration of the glutaraldehyde aqueous solution is 1-3%.

[0025] By adopting the above technical solution and further limiting the concentration of aqueous solutions of hyaluronic acid, elastin-like protein, and glutaraldehyde, a tissue filler with better elasticity and longer effective filling time is obtained. The combination of hyaluronic acid, elastin-like protein, and glutaraldehyde has a better filling effect.

[0026] Preferably, the mass ratio of the hyaluronic acid aqueous solution to the elastin-like aqueous solution is 1:1-4.

[0027] By adopting the above technical solution, the mass ratio of hyaluronic acid aqueous solution to elastin-like aqueous solution is further limited, so that the combination of hyaluronic acid aqueous solution and elastin-like aqueous solution has good solution viscosity and mechanical stability. After being mixed with glutaraldehyde, the resulting tissue filler has good mechanical properties.

[0028] Preferably, the mass ratio of the mixture to the glutaraldehyde aqueous solution is 1:0.5-20.

[0029] By adopting the above technical solution and further limiting the mass ratio of the mixture to the glutaraldehyde aqueous solution, the prepared tissue filler has better mechanical properties and elasticity. Hyaluronic acid, elastin-like protein and glutaraldehyde work together to form a gel network structure, which allows the filler to have a longer filling time, thus helping to fill the skin.

[0030] Preferably, the stirring rate in steps (1), (2), (3) and (4) is 800-900 rpm.

[0031] By adopting the above technical solution and further limiting the stirring rate of each step, a uniformly dispersed aqueous solution is obtained, which makes the final tissue filler have better mechanical stability and has a longer effective filling time when applied to the skin.

[0032] Preferably, the particle size of the composite microspheres is 30-50 μm.

[0033] Preferably, the mass ratio of chitosan, nano-SiO2 and carbon nanotubes is 1:0.2-0.5:0.06-0.09.

[0034] By adopting the above technical solution, the mass ratio of chitosan, nano-SiO2, and carbon nanotubes is further limited within a certain range, resulting in composite microspheres with better elasticity. Carbon nanotubes are loaded on the surface of nano-SiO2, and chitosan coats the nano-SiO2 loaded with carbon nanotubes, thereby increasing the connectivity between nano-SiO2 and carbon nanotubes, improving the stability of nano-SiO2, and helping to improve the stability of composite microspheres in the future.

[0035] Secondly, this application also provides a method for preparing a tissue filler based on elastin, and the application of the tissue filler in medical aesthetic materials.

[0036] In summary, this application has the following beneficial effects:

[0037] 1. In this application, hyaluronic acid and elastin are miscible and mix, which improves the viscosity of the collagen solution. Then, it cross-links with glutaraldehyde to form a gel that is softer than the gel formed by elastin and glutaraldehyde alone. After filling, there is no foreign body sensation. Moreover, the gel is stable under acidic conditions and has good bioactivity. The cross-linking between hyaluronic acid, elastin and glutaraldehyde results in a filler that can supply cell nutrition and promote fibroblast regeneration for a longer period of time. After solidification, it has a longer effect and prolongs the maintenance time. The effective filling time of the filler is more than half a year.

[0038] 2. In this application, hyaluronic acid, elastin-like protein, and glutaraldehyde are dispersed in ultrapure water, and the resulting aqueous solution has good dispersibility, uniformity, and stability. Then, the aqueous solution of hyaluronic acid and the aqueous solution of elastin-like protein are mixed to improve the viscosity of the collagen solution, and the resulting aqueous solution has good stability. When cross-linked with glutaraldehyde, the resulting gel is softer than the gel formed by elastin-like protein and glutaraldehyde alone, has better mechanical properties, no foreign body sensation after filling, and is stable under acidic conditions, with a longer effective filling time.

[0039] 3. This application further limits the concentration of aqueous solutions of hyaluronic acid, elastin-like protein, and glutaraldehyde to obtain a tissue filler with better elasticity and longer effective filling time. The combination of hyaluronic acid, elastin-like protein, and glutaraldehyde has a better filling effect. Attached Figure Description

[0040] Figure 1 It is an amino acid sequence similar to elastin. Detailed Implementation

[0041] The present application will be further described in detail below with reference to the embodiments.

[0042] The raw materials used in the examples and comparative examples are all commercially available. Example

[0043] Example 1

[0044] A method for preparing an elastin-based tissue filler includes the following steps:

[0045] (1) Disperse 3g of hyaluronic acid in ultrapure water and stir until homogeneous to obtain an aqueous solution of hyaluronic acid;

[0046] (2) Disperse 300 mg of elastin in ultrapure water and stir until homogeneous to obtain an elastin aqueous solution;

[0047] (3) Disperse 2g of glutaraldehyde in ultrapure water and stir until homogeneous to obtain an aqueous solution of glutaraldehyde;

[0048] (4) Mix 1 kg of hyaluronic acid aqueous solution from step (1) and 2.5 kg of elastin-like aqueous solution from step (2) for 3 hours to obtain a mixture. Then add 10 kg of glutaraldehyde aqueous solution from step (3) to the mixture and continue stirring for 15 minutes. Add 6 g of composite microspheres and stir for 32 minutes to obtain the tissue filler. The particle size of the composite microspheres is 30-50 μm.

[0049] The preparation method of composite microspheres includes the following steps:

[0050] (1) Disperse 1 kg of chitosan in 2 L of acetic acid solution with a mass fraction of 20%, stir at 65 °C for 50 min, then add 0.2 kg of sodium dodecyl sulfate, and continue stirring for 2 h to obtain a mixture;

[0051] (2) Disperse nano-SiO2 in 2.5L of 10% sodium hydroxide solution, stir at 85℃ for 2h, wash with water, then disperse in 3L of anhydrous ethanol, add carbon nanotubes, sonicate for 3h, and dry to obtain a mixture;

[0052] (3) Spray the mixture from step (1) onto the surface of the mixture from step (2) and dry it at 95°C for 3 hours to obtain composite microspheres with a coating thickness of 0.1 mm.

[0053] The mass ratio of chitosan, nano-SiO2, and carbon nanotubes is 1:0.2:0.09.

[0054] The concentration of the hyaluronic acid aqueous solution is 3 mg / mL, the concentration of the elastin-like aqueous solution is 300 mg / L, and the concentration of the glutaraldehyde aqueous solution is 2%.

[0055] The mass ratio of hyaluronic acid aqueous solution to elastin-like aqueous solution is 1:2.5.

[0056] The mass ratio of the mixed solution to the glutaraldehyde aqueous solution is 1:10.

[0057] The stirring speed in steps (1), (2), (3) and (4) is 850 rpm.

[0058] All elastin-like proteins used were synthesized by Beijing Shunxing Biotechnology Co., Ltd. The amino acid sequences of the elastin-like proteins are shown below. Figure 1 The underlined part is the repeating unit [(VPGKG)9], and the bold part is the spacer (VPGVG).

[0059] Example 2

[0060] A method for preparing an elastin-based tissue filler includes the following steps:

[0061] (1) Disperse 5g of hyaluronic acid in ultrapure water and stir until homogeneous to obtain an aqueous solution of hyaluronic acid;

[0062] (2) Disperse 650 mg of elastin in ultrapure water and stir until homogeneous to obtain an elastin aqueous solution;

[0063] (3) Disperse 3g of glutaraldehyde in ultrapure water and stir until homogeneous to obtain an aqueous solution of glutaraldehyde;

[0064] (4) Mix 1 kg of hyaluronic acid aqueous solution from step (1) and 4 kg of elastin-like aqueous solution from step (2) for 3 hours to obtain a mixture. Then add 20 kg of glutaraldehyde aqueous solution from step (3) to the mixture and continue stirring for 20 minutes. Add 6 g of composite microspheres and stir for 30 minutes to obtain the tissue filler.

[0065] The concentration of the hyaluronic acid aqueous solution is 5 mg / mL, the concentration of the elastin-like aqueous solution is 650 mg / L, and the concentration of the glutaraldehyde aqueous solution is 3%.

[0066] The mass ratio of hyaluronic acid aqueous solution to elastin-like aqueous solution is 1:4.

[0067] The mass ratio of the mixture to the glutaraldehyde aqueous solution is 1:20.

[0068] The stirring speed in steps (1), (2), (3) and (4) is 900 rpm.

[0069] Example 3

[0070] A method for preparing an elastin-based tissue filler includes the following steps:

[0071] (1) Disperse 1g of hyaluronic acid in ultrapure water and stir until homogeneous to obtain an aqueous solution of hyaluronic acid;

[0072] (2) Disperse 50 mg of elastin in ultrapure water and stir until homogeneous to obtain an elastin aqueous solution;

[0073] (3) Disperse 1g of glutaraldehyde in ultrapure water and stir until homogeneous to obtain an aqueous solution of glutaraldehyde;

[0074] (4) Mix 1 kg of hyaluronic acid aqueous solution from step (1) and 1 kg of elastin-like aqueous solution from step (2) for 2 hours to obtain a mixture. Then add 1 kg of glutaraldehyde aqueous solution from step (3) to the mixture and continue stirring for 5 minutes. Add 6 g of composite microspheres and stir for 35 minutes to obtain the tissue filler.

[0075] The concentration of the hyaluronic acid aqueous solution is 1 mg / mL, the concentration of the elastin-like aqueous solution is 50 mg / L, and the concentration of the glutaraldehyde aqueous solution is 1%.

[0076] The mass ratio of hyaluronic acid aqueous solution to elastin-like aqueous solution is 1:1.

[0077] The mass ratio of the mixture to the glutaraldehyde aqueous solution is 1:0.5.

[0078] The stirring speed in steps (1), (2), (3) and (4) is 800 rpm.

[0079] Example 4

[0080] A method for preparing a tissue filler based on elastin-like protein differs from Example 1 in that hyaluronic acid, elastin-like protein, glutaraldehyde, and composite microspheres are mixed, dispersed in ultrapure water, and stirred for 4 hours to obtain the tissue filler.

[0081] Example 5

[0082] A method for preparing an elastin-based tissue filler differs from Example 1 in that the concentration of the hyaluronic acid aqueous solution is 0.5 mg / mL.

[0083] Example 6

[0084] A method for preparing an elastin-based tissue filler differs from Example 1 in that the concentration of the hyaluronic acid aqueous solution is 7 mg / mL.

[0085] Example 7

[0086] A method for preparing an elastin-based tissue filler differs from Example 1 in that the concentration of the elastin-based aqueous solution is 30 mg / L.

[0087] Example 8

[0088] A method for preparing an elastin-based tissue filler differs from Example 1 in that the concentration of the elastin-based aqueous solution is 700 mg / L.

[0089] Example 9

[0090] A method for preparing an elastin-based tissue filler differs from Example 1 in that the concentration of the glutaraldehyde aqueous solution is 0.2%.

[0091] Example 10

[0092] A method for preparing an elastin-based tissue filler differs from Example 1 in that the concentration of the glutaraldehyde aqueous solution is 4%.

[0093] Example 11

[0094] A method for preparing a tissue filler based on elastin, which differs from Example 1 in that the mass ratio of hyaluronic acid aqueous solution to elastin aqueous solution is 1:0.2.

[0095] Example 12

[0096] A method for preparing an elastin-based tissue filler differs from Example 1 in that the mass ratio of hyaluronic acid aqueous solution to elastin-based aqueous solution is 1:6.

[0097] Example 13

[0098] A method for preparing a tissue filler based on elastin differs from Example 1 in that the mass ratio of the mixture to the glutaraldehyde aqueous solution is 1:0.1.

[0099] Example 14

[0100] A method for preparing an elastin-based tissue filler differs from Example 1 in that the mass ratio of the mixture to the glutaraldehyde aqueous solution is 1:25.

[0101] Example 15

[0102] A method for preparing an elastin-based tissue filler differs from Example 15 in that the mass ratio of chitosan, nano-SiO2, and carbon nanotubes in the method for preparing the composite microspheres is 1:0.5:0.06.

[0103] Example 16

[0104] A method for preparing an elastin-based tissue filler differs from Example 15 in that the mass ratio of chitosan, nano-SiO2, and carbon nanotubes is 1:0.8:0.01.

[0105] Comparative Example 1

[0106] A method for preparing an elastin-based tissue filler differs from Example 1 in that hyaluronic acid is not added.

[0107] Comparative Example 2

[0108] A method for preparing an elastin-based tissue filler differs from Example 1 in that no elastin is added.

[0109] Comparative Example 3

[0110] A method for preparing an elastin-based tissue filler differs from Example 15 in that the method for preparing the composite microspheres does not involve the addition of nano-SiO2.

[0111] Comparative Example 4

[0112] A method for preparing an elastin-based tissue filler differs from Example 15 in that carbon nanotubes are not added in the method for preparing the composite microspheres.

[0113] Comparative Example 5

[0114] A method for preparing an elastin-based tissue filler differs from Example 15 in that chitosan is not added in the preparation method of the composite microspheres.

[0115] The tissue fillers prepared in Examples 1-16 and Comparative Examples 1-5 were subjected to performance tests.

[0116] Degradation rate test: Experimental animals (SD rats) were anesthetized by intraperitoneal injection of 3% pentobarbital (30.0 mg / kg), and the skin on the back was disinfected with ethanol. The tissue filler prepared in the examples and comparative examples was injected into the subcutaneous tissue of the rat back using a 1 mL syringe, with an injection dose of 400 μL. At 3, 6, 8 and 12 months after injection, the subcutaneous tissue and surrounding tissue of each group of animals were randomly collected under aseptic conditions from the injection site in three batches. The tissue was fixed in 4% paraformaldehyde. After 1 week, the tissue sections were dehydrated by gradient ethanol, cleared with xylene, and embedded in paraffin. Then, the sections were dewaxed and rehydrated, stained with Harris hematoxylin, separated by 0.5% hydrochloric acid ethanol, stained with 95% ethanol eosin, dehydrated by gradient ethanol, cleared with xylene, and fixed with neutral resin. The absorption and degradation of the tissue filler at the injection site were observed under a microscope.

[0117] Viscosity modulus and elastic modulus testing: The tissue fillers prepared in the examples and comparative examples were subjected to relevant performance tests. The viscosity modulus and elastic modulus were tested using an Anton Paar rotational rheometer (MCR301). The test conditions were as follows: the sample was heated to room temperature (25°C) and then held at that temperature for 30 min. The sample was scanned at a frequency of 0.1 Hz under a strain of 0.1%. The results are shown in Table 1.

[0118] Table 1 Test data for the examples and comparative examples

[0119]

[0120] As shown in Table 1, the tissue fillers prepared in Examples 1-3 and Example 15 of this application exhibit good elasticity and effective filling time. Specifically, Example 1 showed a degradation rate of 10% after 3 months, 30% after 6 months, 50% after 8 months, and 80% after 12 months, with a viscous modulus of 30.8 Pa and an elastic modulus of 325.6 Pa. This indicates that the miscibility and mixing of hyaluronic acid and elastin-like proteins improve the viscosity of the collagen solution. Further cross-linking with glutaraldehyde and then combining with composite microspheres results in a gel with good elasticity and a longer effective filling time.

[0121] In Example 4, the preparation method of the tissue filler involved mixing hyaluronic acid, elastin-like substances, glutaraldehyde, and ultrapure water together without step-by-step mixing. Table 1 shows that the degradation rate was 20% after 3 months, 40% after 6 months, 61% after 8 months, and 91% after 12 months. The viscous modulus was 39.7 Pa, and the elastic modulus was 300.1 Pa. This indicates that dispersing hyaluronic acid, elastin-like substances, glutaraldehyde, and composite microspheres in ultrapure water separately yields an aqueous solution with good dispersibility, uniformity, and stability. The resulting tissue filler exhibits superior elasticity and effective filling time.

[0122] Examples 5-6 involved varying the concentration of the hyaluronic acid aqueous solution. Table 1 shows that in Example 5, the degradation rate was 16% after 3 months, 35% after 6 months, 58% after 8 months, and 87% after 12 months, with a viscous modulus of 36.4 Pa and an elastic modulus of 310.2 Pa. This indicates that further limiting the concentration of the hyaluronic acid aqueous solution yields a tissue filler with better elasticity and a longer effective filling time. The combination of hyaluronic acid aqueous solution and elastin-like and glutaraldehyde aqueous solutions provides a better filling effect.

[0123] Examples 7-8 varied the concentration of the elastin-like aqueous solution. As shown in Table 1, in Example 7, the degradation rate was 18% after 3 months of injection, 39% after 6 months, 59% after 8 months, and 89% after 12 months. The viscous modulus was 37.5 Pa, and the elastic modulus was 305.6 Pa. This indicates that further limiting the concentration of the elastin-like aqueous solution yielded a tissue filler with better elasticity and a longer effective filling time. The elastin-like aqueous solution combined with the aqueous solutions of hyaluronic acid and glutaraldehyde had a good filling effect.

[0124] Examples 9-10 involved varying the concentration of glutaraldehyde aqueous solution. Table 1 shows that in Example 9, the degradation rate was 15% after 3 months, 33% after 6 months, 56% after 8 months, and 86% after 12 months, with a viscous modulus of 35.9 Pa and an elastic modulus of 311.9 Pa. This indicates that further limiting the concentration of the glutaraldehyde aqueous solution yields a tissue filler with better elasticity and a longer effective filling time. The combination of glutaraldehyde aqueous solution and aqueous solutions of elastin-like proteins and hyaluronic acid also demonstrates a good filling effect.

[0125] Examples 11-12 varied the mass ratio of hyaluronic acid aqueous solution to elastin-like aqueous solution. Table 1 shows that in Example 11, the degradation rate was 22% after 3 months, 43% after 6 months, 64% after 8 months, and 93% after 12 months, with a viscosity modulus of 45.0 Pa and an elastic modulus of 293.4 Pa. This indicates that further limiting the mass ratio of hyaluronic acid aqueous solution to elastin-like aqueous solution resulted in a solution with better viscosity and mechanical stability. Subsequent mixing with glutaraldehyde yielded a tissue filler with better mechanical properties.

[0126] Examples 13-14 varied the mass ratio of the mixture to the glutaraldehyde aqueous solution. Table 1 shows that in Example 13, the degradation rate was 23% after 3 months, 45% after 6 months, 66% after 8 months, and 95% after 12 months, with a viscous modulus of 45.6 Pa and an elastic modulus of 291.0 Pa. This indicates that further limiting the mass ratio of the mixture to the glutaraldehyde aqueous solution results in a tissue filler with superior mechanical properties and elasticity. The hyaluronic acid, elastin-like proteins, and glutaraldehyde work together to form a gel network structure, allowing for a longer filling time and thus contributing to skin filling.

[0127] Example 16: The mass ratio of chitosan, nano-SiO2, and carbon nanotubes was varied. Table 1 shows that the degradation rate was 15% after 3 months of injection, 34% after 6 months, 55% after 8 months, and 86% after 12 months. The viscous modulus was 33.1 Pa, and the elastic modulus was 310.4 Pa. This indicates that within a certain range, the mass ratio of chitosan, nano-SiO2, and carbon nanotubes yields composite microspheres with good elasticity. The carbon nanotubes are loaded on the surface of the nano-SiO2, and the chitosan coats the carbon nanotube-loaded nano-SiO2, thereby increasing the connectivity between the nano-SiO2 and carbon nanotubes and improving the stability of the nano-SiO2, which helps to improve the stability of the composite microspheres in the future. Comparative Example 1: No hyaluronic acid was added. Table 1 shows that the degradation rate was 40% after 3 months of injection, 80% after 6 months, and 100% after both 8 and 12 months. The viscous modulus was 25.6 Pa, and the elastic modulus was 335.1 Pa. The results indicate that only tissue fillers prepared from elastin-like proteins and glutaraldehyde have a relatively fast degradation rate, completely degrading within 8 months, while also exhibiting good elasticity.

[0128] Comparative Example 2, without the addition of elastin-like substances, showed a degradation rate of 60% at 3 months after injection, and 100% at 6, 8, and 12 months. The viscous modulus was 65.1 Pa, and the elastic modulus was 250.3 Pa. This indicates that only the tissue filler prepared with hyaluronic acid and glutaraldehyde exhibited a relatively rapid degradation rate, complete degradation at 6 months, high viscosity, but decreased elasticity.

[0129] In Comparative Example 3, the composite microspheres were prepared without the addition of nano-SiO2. Table 1 shows that the degradation rate was 30% after 3 months, 50% after 6 months, 70% after 8 months, and 100% after 12 months, with a viscous modulus of 52.3 Pa and an elastic modulus of 280.1 Pa. This indicates that nano-SiO2, as a bioactive glass, when injected into the skin, can repair, replace, and regenerate body tissues, and can form bonds between tissues and materials. The degradation products of nano-SiO2 can promote the production of skin growth factors, promote cell proliferation, enhance osteoblast gene expression, and promote bone tissue growth.

[0130] In Comparative Example 4, no carbon nanotubes were added during the preparation of the composite microspheres. Table 1 shows that the degradation rate was 35% after 3 months of injection, 54% after 6 months, 76% after 8 months, and 100% after 12 months. The viscous modulus was 56.8 Pa, and the elastic modulus was 270.1 Pa. This indicates that carbon nanotubes are loaded onto the surface and within the pores of nano-SiO2, thereby improving the mechanical properties and supporting performance of nano-SiO2, and subsequently enhancing the elasticity of the composite microspheres.

[0131] In Comparative Example 5, chitosan was not added during the preparation of the composite microspheres. Table 1 shows that the degradation rate was 28% after 3 months of injection, 49% after 6 months, 67% after 8 months, and 100% after 12 months. The viscous modulus was 50.1 Pa, and the elastic modulus was 283.1 Pa. This indicates that spraying the mixture onto the surface of the mixture, allowing the mixture to coat the mixture, increases the connectivity between nano-SiO2 and carbon nanotubes. This ensures that the carbon nanotubes are firmly loaded on the surface of the nano-SiO2, thereby improving the mechanical stability of the composite microspheres. This is beneficial for subsequent application in the skin, improving the stability of the composite microspheres in the skin, and thus prolonging the effective filling time of the tissue filler.

[0132] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing a tissue filler based on elastin, characterized in that, Includes the following steps: (1) Disperse hyaluronic acid in ultrapure water and stir until homogeneous to obtain an aqueous solution of hyaluronic acid; (2) Disperse the elastin-like protein in ultrapure water and stir until homogeneous to obtain an elastin-like protein aqueous solution; (3) Disperse glutaraldehyde in ultrapure water and stir until homogeneous to obtain an aqueous solution of glutaraldehyde; (4) Mix the hyaluronic acid aqueous solution from step (1) and the elastin-like aqueous solution from step (2) for 2-3 hours to obtain a mixture. Then add the glutaraldehyde aqueous solution from step (3) to the mixture and continue stirring for 5-20 minutes. Add the composite microspheres and stir for 30-35 minutes to obtain the tissue filler. The method for preparing the composite microspheres includes the following steps: (a) Disperse chitosan in acetic acid solution and stir at 60-65℃ for 30-50 min, then add sodium dodecyl sulfate and continue stirring to obtain a mixture; (b) Disperse nano-SiO2 in sodium hydroxide solution, stir at 80-85℃ for 1-2 h, wash with water, then disperse in anhydrous ethanol, add carbon nanotubes, sonicate for 2-3 h, and dry to obtain a mixture; (c) Spray the mixture from step (a) onto the surface of the mixture from step (b) and dry it at 90-95°C for 2-3 hours to obtain composite microspheres.

2. The method for preparing a tissue filler based on elastin according to claim 1, characterized in that, The concentration of the hyaluronic acid aqueous solution is 1-5 mg / mL, the concentration of the elastin-like aqueous solution is 50-650 mg / L, and the concentration of the glutaraldehyde aqueous solution is 1-3%.

3. The method for preparing a tissue filler based on elastin according to claim 1, characterized in that, The mass ratio of the hyaluronic acid aqueous solution to the elastin-like aqueous solution is 1:1-4.

4. A method for preparing a tissue filler based on elastin according to claim 1, characterized in that, The mass ratio of the mixture to the glutaraldehyde aqueous solution is 1:0.5-20.

5. The method for preparing a tissue filler based on elastin according to claim 1, characterized in that, The stirring rate in steps (1), (2), (3) and (4) is 800-900 rpm.

6. The method for preparing a tissue filler based on elastin according to claim 1, characterized in that, The composite microspheres have a particle size of 30-50 μm.

7. The method for preparing a tissue filler based on elastin according to claim 1, characterized in that, The mass ratio of chitosan, nano-SiO2, and carbon nanotubes is 1:0.2-0.5:0.06-0.

09.

8. The application of the tissue filler prepared by the method of preparing an elastin-based tissue filler according to any one of claims 1-7 in medical aesthetic materials.

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