Preparation method of compound collagen filler for injection

Abstract

The invention relates to a preparation method of a collagen filling agent for injection, which is characterized in that non-crosslinked collagen, crosslinked collagen and degradable polymer microspheres are compounded and mixed according to a certain proportion to achieve an instant filling effect, and the degradable polymer microspheres play a long-term role. Amino acid components degraded by the collagen in the first and middle stages can become nutrient substances for stimulating the polyester microspheres to generate collagen regeneration. The cross-linked collagen disclosed by the invention is relatively high in yield and less in water loss through a gradient irradiation cross-linking technology. The non-crosslinked collagen gel can provide a nutritional repair microenvironment and a filling effect in the earlier stage, the crosslinked extracellular matrix can provide a repair microenvironment in the middle stage of filling, and degradation of the polyester microspheres in the later stage promotes collagen regeneration, so that the skin has a good microenvironment in the whole repair period, and the skin elasticity is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical aesthetic injections, and specifically relates to a method for preparing a composite collagen filler for injection. Background Art

[0002] In terms of the selection of raw materials for injectable fillers, sodium hyaluronate and collagen are still the main ones. However, this type of material is easily absorbed by the body's tissues, has a short degradation cycle, and the filling effect is to temporarily occupy the skin volume and cause limited local host tissue reaction (limited regenerative fibrosis). It is difficult to maintain long-term filling and inducing collagen regeneration effects. The toxic cross-linking agent components introduced to provide long-term degradation time also limit its application.

[0003] Due to these problems, several products using biodegradable polymers for tissue repair treatment have been developed recently, and these products have been developed and used as filler preparations for existing biocompatible polymers, or preparations that are dispersed into microparticles through a viscous medium (CMC, etc.) after treating water-insoluble polymers, such as polycaprolactone fillers, etc. This type of material promotes the regeneration of collagen through the stimulation of degradation products to play a repairing role. Since degradation requires a certain amount of time, the immediate effect is not obvious, and the nutritional effect of CMC is not strong. Collagen used alone has poor physical and mechanical properties, single performance, and due to its strong hydrophilicity, it is easily degraded by collagenase in the body, which limits its application. Although collagen cross-linked with chemical agents can increase the degradation time of the gel, it has certain toxicity; polyester fillers cannot achieve immediate stimulation of collagen, and the use of adhesive CMC gel has a filling effect, but it is not a human component and does not have a nutritional regeneration effect; hyaluronic acid gel will also have residual toxic effects after cross-linking.

[0004] CN118649289A discloses a collagen implant, which sequentially performs physical crosslinking, chemical crosslinking and radiation crosslinking on collagen to obtain collagen fibers, and then disperses and emulsifies the solution to obtain a collagen implant. In order to increase the degree of crosslinking, the patent uses three crosslinking methods. The triple crosslinking method enables the product to have a high degree of crosslinking and a low enzymatic hydrolysis rate, giving the filler a more durable filling effect. However, chemical crosslinking inevitably introduces chemical crosslinking agents, such as dialdehydes, carbodiimides, etc. The residues of the crosslinking agents bring more sensitization risks and even teratogenic risks to clinical use.

[0005] CN117618671A discloses a collagen filler with an average pore size D50 of 50 μm to 500 μm, a porosity of 80%-99.5%, a cross-linking degree of 10% to 70%, and an in vitro enzymatic loss rate of less than 40%. The patent controls the relative spacing of collagen fiber bundles during the cross-linking process by controlling the pore size using microspheres as templates, thereby controlling the size of the holes after the collagen fiber bundles form a cross-linked skeleton; on the other hand, by controlling mechanical stirring and microsphere addition methods, a dynamic single-template (or multi-template) cross-linking process is achieved, so that each collagen fiber bundle can obtain the same cross-linking opportunity, thereby achieving a more thorough and uniform cross-linking effect of the collagen implant.

[0006] CN117582549A discloses an injectable recombinant collagen soft tissue filling gel, which comprises the following raw materials in weight percentage: 5%-10% of type III recombinant collagen solution, 2%-3% of sodium hyaluronate solution for injection, 1%-3% of medicinal glycerol, 1%-5% of medical hydroxyapatite, and the balance is water. The injectable recombinant collagen soft tissue gel prepared by mixing type III recombinant collagen solution, sodium hyaluronate solution for injection, medicinal glycerol and medical hydroxyapatite uses the screened core functional region fragments with high activity and high water solubility, which are 100% identical to the corresponding fragments of human collagen, eliminates immunogenicity, and has the characteristics of high biocompatibility, high biological activity, high water solubility and no viral reaction.

[0007] However, the above-mentioned prior art includes products that are a composite of collagen and microspheres for injection filling, but they cannot achieve a long-term filling effect and the risk of collagen allergy is relatively high. Summary of the invention

[0008] In order to solve the defects of short action time, poor filling effect and short duration in fillers containing collagen, the present invention proposes an injectable collagen filler, which increases the filling strength and duration by cross-linking collagen and adding injectable filling microspheres. Specifically, the present invention provides the following technical solutions to achieve the above purpose:

[0009] A method for preparing an injectable collagen filler comprises the following steps:

[0010] (S1) The extracted collagen solution is subjected to salting-out precipitation, and the precipitate is dialyzed and freeze-dried to obtain collagen;

[0011] (S2) dissolving collagen with hydrochloric acid and then homogenizing with PBS buffer solution to prepare a non-cross-linked collagen gel;

[0012] (S3) Dissolving collagen with hydrochloric acid and then homogenizing with PBS buffer solution, irradiating the obtained collagen gel by gradient irradiation cross-linking, and granulating with a mesh after irradiation to obtain a cross-linked collagen gel; the gradient irradiation cross-linking is that the irradiation intensity is gradually increased from 10-15 kgy to 25-30 kgy, and then the irradiation intensity is maintained;

[0013] (S4) Mixing the non-cross-linked collagen gel obtained in step (S2), the cross-linked collagen gel obtained in step (S3), and the microspheres for injection filling, degassing and filling, and obtaining an injectable collagen filler.

[0014] Furthermore, in step (S1), the source of collagen includes but is not limited to animal root tendons, such as bovine tendons and porcine tendons; animal skins, such as cowhide and porcine skin; bovine pericardium and arachnoid membrane.

[0015] Furthermore, when preparing non-cross-linked collagen in (S2), the collagen is derived from animal skins such as cowhide and pig skin; when preparing non-cross-linked collagen in step (S3), the collagen is derived from animal tendons such as bovine Achilles tendon and pig Achilles tendon, preferably bovine Achilles tendon. Bovine Achilles tendon collagen is denser, and the strength of the cross-linked collagen is higher, and the collagen content is also higher, which is more suitable as a raw material for cross-linked collagen. The inventors have found that the non-cross-linked collagen is prepared with collagen derived from animal skins, and the cross-linked collagen is prepared with collagen derived from animal Achilles tendons, and the resulting collagen filler has the best filling effect.

[0016] Furthermore, the concentration of collagen in the non-cross-linked collagen gel is 20-35 mg / mL, and the concentration of collagen in the cross-linked collagen gel is 25-32 mg / mL. The concentration limit of collagen in the cross-linked collagen gel should be more stringent. A higher concentration can increase the yield of the product after water loss and the yield; however, the concentration cannot be too high, otherwise the strength is too high, granulation is difficult, and the formed filler pushing force is also large, which is inconvenient to use.

[0017] Furthermore, the extraction process of collagen is well known in the art. For example, for collagen derived from animal Achilles tendon, the fascia of the animal Achilles tendon is removed, and the tendon is washed, frozen, sliced, and enzymatically hydrolyzed in a pepsin solution. The enzymatic hydrolyzate is centrifuged and the supernatant is salted out to obtain collagen derived from animal Achilles tendon. For collagen derived from animal skin, the hair of the animal skin is removed, the fat layer and the epidermis are removed, leaving the dermis, washed with water, frozen for storage, immersed in a 4-8°C Tris-NaCl buffer solution, washed with water, enzymatically hydrolyzed in a pepsin solution, centrifuged to remove the residue, and salted out to obtain collagen derived from animal skin.

[0018] Furthermore, when extracting collagen from animal Achilles tendon, the freezing temperature is -15°C to 0°C, the slice thickness is 0.5-2mm, the pH of the pepsin solution is 2-4, the usage ratio of animal Achilles tendon slices to the pepsin solution is 1kg:100-200L; the concentration of the pepsin solution is 1g / 10L to 1g / 30L, the enzymatic hydrolysis time is 3-7 days, the salting out uses a saturated NaCl solution, the precipitated precipitate is dialyzed with pH=2-3, pH=3-4 and purified water for 7-12 days, and then freeze-dried to obtain the collagen from animal Achilles tendon; source When extracting collagen from animal skin, the pH of the Tris-NaCl buffer solution is 7.5-8.5, the concentration of animal skin in the pepsin solution is 1-5 g / L, the enzymatic hydrolysis time is 2-5 days, the flocculent precipitate obtained by salting out is redissolved in an acetic acid solution (acetic acid concentration is 0.1-0.5 mol / L), the insoluble particles are removed by centrifugation, the salting out and redissolution steps are repeated 2-5 times, the precipitate obtained by the last salting out is dialyzed in a 0.1-0.2 M acetic acid solution for 2-4 days, and then dialyzed in purified water for 1-2 days, and freeze-dried to obtain collagen from animal skin.

[0019] Furthermore, in step (S2) and step (S3), the pH of the hydrochloric acid is 3-4; the pH of the PBS buffer solution is 7.2-7.6, such as pH=7.3, pH=7.4, pH=7.5.

[0020] Furthermore, in step (S2), the ratio of collagen, hydrochloric acid, and PBS buffer is 1 g: 20-30 mL: 2-3 mL.

[0021] Furthermore, in step (S3), the time of gradient irradiation is 15-30min, and then the irradiation intensity is maintained for 3-5min. The intensity of gradient irradiation gradually increases in a nearly linear manner, for example, the irradiation intensity gradually increases from 10kgy to 30ky within 20min, and the irradiation intensity increase rate is about 1kgy / min; for example, the irradiation intensity gradually increases from 15kgy to 25ky within 20min, and the irradiation intensity increase rate is about 0.5kgy / min. After irradiation, the sieve is granulated, and the sieve aperture is 90-150μm.

[0022] Further, in step (S4), the microspheres for injection filling are well known in the art, including but not limited to PCL microspheres, PLLA microspheres, PLGA microspheres, and the particle size of the microspheres for injection filling is 30-50 μm. The microspheres for injection filling can be purchased commercially or homemade, and their preparation methods are well known in the art. For example, for PCL microspheres, the PCL polymer is dissolved in dichloromethane and / or chloroform to prepare a 10-40wt% polymer oil phase solution, and PVA is dissolved at 70-90°C to prepare a 0.2-2wt% PVA aqueous solution, high-speed shearing, the volume ratio of the oil phase to the water phase is 1:2-10, the dichloromethane / chloroform solvent is volatilized by stirring, and sieved to obtain PCL microspheres with a particle size of 30-50 μm.

[0023] Furthermore, in step (S4), the mass ratio of the non-cross-linked collagen gel, the cross-linked collagen gel, and the microspheres for injection filling is 2-4:3-5:1-2.

[0024] The present invention uses non-cross-linked collagen, cross-linked collagen, and degradable polymer microspheres for injection to be compounded and mixed in a certain proportion, so that an immediate filling effect can be achieved, and the polymer microspheres play a long-term role. The amino acid components of the collagen degradation in the early and middle stages can become nutrients that stimulate the polyester microspheres to produce collagen regeneration. However, the biggest problem with collagen for medical beauty injection filling is the short duration and allergy. The present invention obtains cross-linked collagen with different cross-linking degrees through different irradiation intensities and compounded with non-cross-linked collagen to achieve the purpose of long filling duration and good filling effect; the collagen allergens of the present invention are less extracted from collagen than other materials, reducing allergies. The cross-linked collagen of the present invention is cross-linked by irradiation technology without adding any chemical cross-linking agent, and there is no chemical toxicity. Gradient irradiation with gradually increasing irradiation intensity can have a higher yield and less water loss at the same concentration. The non-cross-linked collagen gel can provide a nutritional repair microenvironment and filling effect in the early stage, the cross-linked extracellular matrix can provide a repair microenvironment in the middle stage of filling, and the degradation of polyester microspheres in the later stage promotes collagen regeneration, so that the skin has a good microenvironment throughout the repair cycle and increases skin elasticity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a photograph of the cross-linked collagen gel obtained in step S3 of Example 1.

[0026] Figure 2 This is a photograph of the cross-linked collagen gel obtained in Comparative Example 1.

[0027] Figure 3 This is a photograph of the cross-linked collagen gel obtained in Comparative Example 2.

[0028] Figure 4This is a photo of the stained slices after the filler of Example 1 was implanted subcutaneously in rabbits for 12 months.

[0029] Figure 5 This is a stained section photograph of the filler of Example 1 after subcutaneous implantation in rabbits for 12 months.

[0030] Figure 6 This is a stained section photograph of the filler of Example 3 after subcutaneous implantation in rabbits for 12 months.

[0031] Figure 7 This is a stained section photograph of the filler of Example 3 after subcutaneous implantation in rabbits for 12 months. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further explained below through specific embodiments.

[0033] Example 1

[0034] (S1) Extraction of collagen:

[0035] (1.1) The forked part of the bovine Achilles tendon is cut into a Y shape, and the main trunk is retained. The fascia of the bovine Achilles tendon is then removed, and the tendon is washed with purified water for no less than 3 times, drained, and frozen in a refrigerator at a temperature of -15 to 0°C. The cross-section slices (thickness 0.5-2 mm) are taken out from the refrigerator, and the slices are added to a pepsin solution of pH = 2 to 4 (bovine Achilles tendon slices: enzymatic solution = 1kg / 100L to 1kg / 200kg), the enzymatic solution concentration is (1g / 10L to 1g / 30L), and the enzymatic solution time is 3-7 days. The supernatant is then collected by centrifugation, and the supernatant is salted out with saturated sodium chloride. The precipitated white flocs are dialyzed in pH = 3, pH = 4 and purified water for 7 days respectively, and then the dialyzate is freeze-dried to obtain bovine Achilles tendon collagen.

[0036] (1.2) Select fresh pig skin, remove hair and dirt, use a knife to remove the subcutaneous fat layer and epidermis, leaving the dermis close to the epidermis, cut into 2 cm × 2 cm pieces, rinse with deionized water 3 times, and store the treated pig skin at -20°C; soak the pig skin in Tris-NaCl (Tris: 0.05 mol / L; NaCl: 1 mol / L; pH 7.5) buffer solution (solid-liquid ratio 1:20) at 4°C for 12 h, and then rinse with deionized water 3 times; take the defatted pig skin and hydrolyze it in a pepsin solution of 1 / 100,000 at pH 2-3 at room temperature for 48 h. , the pig skin concentration is 1 mg / mL, and the unreacted residue is removed by centrifugation; saturated NaCl is added to the solution after the above reaction, and salting out at 4°C until flocculent precipitates appear in the slurry, and centrifugation is performed (4°C, 10000rpm, 15min), the lower precipitate is collected, and it is redissolved in a solution with a pH value of 2.2 (0.5mol / L acetic acid), and insoluble particles are removed by centrifugation (4°C, 10000rpm, 15min), and this is repeated 3 times; the last salting-out precipitate is placed in a dialysis bag, dialyzed in a 0.1M acetic acid solution for 48h, and then dialyzed in deionized water for 24h, and then vacuum freeze-dried to obtain pig skin collagen.

[0037] (S2) Preparation of non-cross-linked collagen gel: 700 mg of freeze-dried pig skin collagen was dissolved in 18 ml of hydrochloric acid at pH = 3, and then homogenized with 2 ml of PBS to prepare a non-cross-linked collagen gel, wherein the collagen concentration was 35 mg / ml.

[0038] (S3) Preparation of cross-linked collagen: 1400 mg of freeze-dried bovine Achilles tendon collagen was dissolved in 45 ml of hydrochloric acid at pH = 3 and then homogenized with 5 ml of PBS to prepare a collagen gel with a collagen concentration of 28 mg / ml. The gel was cross-linked by cobalt 60 irradiation. The irradiation intensity was increased from 10 kgy to 30 kgy at a rate of 1 kgy / min within 20 min, and then continued to irradiate for 5 min. The gel was granulated using a 150 mesh screen.

[0039] (S4) Preparation of filler: Weigh 100g of polycaprolactone, the molecular weight of polycaprolactone is 25000, the molecular weight distribution coefficient (PDI) is 2.3, dissolve it in 250ml of dichloromethane to prepare a polymer oil phase solution; dissolve 3.2g of PVA raw material in injection water at 70-90°C, cool it, and prepare a 0.4% (W / V) PVA aqueous solution; the oil phase solution and the PVA aqueous solution enter the online shearing machine through a pipeline with a flow pump to mix, shear emulsify at a speed of 3200rpm, then stir to volatilize dichloromethane, use a 30μm sieve and a 50μm sieve, and finally freeze-dry to prepare 30-50um PCL microspheres. Non-cross-linked collagen gel, cross-linked collagen gel, and PCL microspheres are mixed in a mass ratio of 3:5:2, degassed and filled in a prefilled syringe for use.

[0040] Example 2

[0041] Other conditions are the same as those in Example 1, except that the collagen used in step (S2) and step (S3) is pig skin collagen.

[0042] Example 3

[0043] Other conditions are the same as those in Example 1, except that the collagen used in step (S2) and step (S3) is bovine Achilles tendon collagen.

[0044] Example 4

[0045] Other conditions are the same as those in Example 1, except that in step (S2), the collagen concentration of the non-cross-linked collagen gel is 20 mg / mL; in step (S2), the collagen concentration of the cross-linked collagen gel is 32 mg / mL.

[0046] Example 5

[0047] The other conditions were the same as those in Example 1, except that in step (S4), the mass ratio of non-cross-linked collagen gel, cross-linked collagen gel, and PCL microspheres was 4:4:2.

[0048] Example 6

[0049] The other conditions were the same as those in Example 1, except that in step (S4), the mass ratio of non-cross-linked collagen gel, cross-linked collagen gel, and PCL microspheres was 2:3:1.

[0050] Example 7

[0051] The other conditions were the same as those in Example 1, except that in step (S3), the collagen concentration of the cross-linked collagen gel was 25 mg / mL.

[0052] Example 8

[0053] The other conditions are the same as those in Example 1, except that in step (S3), the gradient irradiation is performed by increasing the irradiation intensity from 15 kgy to 25 kgy at a rate of 0.5 kgy / min within 20 min, and then continuing the irradiation for 5 min.

[0054] Comparative Example 1

[0055] The other conditions were the same as those in Example 1, except that in step (S3), the collagen concentration of the cross-linked collagen gel was 20 mg / mL.

[0056] Comparative Example 2

[0057] Other conditions are the same as those in Example 1, except that in step (S3), the irradiation is changed to a fixed irradiation intensity of 20 kgy for 20 min.

[0058] Comparative Example 3

[0059] The other conditions were the same as those in Example 1, except that in step (S4), the filler was a mixture of cross-linked collagen gel and PCL microspheres in a mass ratio of 8:2, that is, no non-cross-linked collagen gel was added.

[0060] Comparative Example 4

[0061] Other conditions are the same as those in Example 1, except that in step (S4), the filler is a mixture of non-cross-linked collagen gel and PCL microspheres in a mass ratio of 8:2, that is, no cross-linked collagen gel is added.

[0062] Application Example 1

[0063] The bovine Achilles tendon collagen and pig skin collagen prepared in step S1 of Example 1 were subjected to terminal peptide detection (ELISA), DNA residue (fluorescence method), and α-antigen detection. The results are shown in Table 1 below.

[0064] Table 1 Residue detection

[0065]

[0066] Analysis: Regarding the severity of allergens, DNA residues>α-antigen>terminal peptide, and the animal-derived collagen obtained by the present invention has a lower immunological response.

[0067] Application Example 2

[0068] In the above embodiments and comparative examples, the mass of the collagen gel before and after cross-linking in step S3 was tested, and the yield after water loss was calculated, and the yield after water loss = the mass of the gel after cross-linking / the mass of the gel before cross-linking. The results are shown in Table 2 below. Figure 1 is a photograph of the cross-linked collagen gel obtained in step S3 of Example 1; Figure 2is a photograph of the cross-linked collagen gel obtained in Comparative Example 1; Figure 3 This is a photograph of the cross-linked collagen gel obtained in Comparative Example 2.

[0069] Table 2 Cross-linked collagen gel yield test

[0070]

[0071]

[0072] It can be seen that the gel strength of Comparative Example 1 after cross-linking at a concentration of 20 mg / ml is weak, it is easy to break in the middle, and the yield after cross-linking is also low (more water loss). After being configured into an injection, the elastic modulus (filling performance) is weak. Comparative Example 1 uses a fixed irradiation intensity, and the yield and elastic modulus also decrease to a certain extent. The cross-linked collagen in Example 1 is not easy to break when pressed and has higher elasticity.

[0073] Application Example 3

[0074] The viscosity and viscoelastic modulus of the fillers obtained in the examples and comparative examples were measured by rheometer, and subcutaneous implantation experiments were performed on rabbits to observe degradation and filling effects. The results are shown in Table 3 below. Degradation performance is based on the presence of residual microspheres and sufficient filling performance. The viscosity of the injectable filler at 0.25Hz is preferably in the range of 200-350Mpa·s. The appropriate viscosity can have both good injectability and filling effect. If the viscosity is too low, the filling effect is average; if the viscosity is too high, injection is difficult and the needle is easily blocked.

[0075] Table 3 Filler effect test

[0076]

[0077]

[0078] Figure 4 This is a stained photograph of a section of the filler of Example 1 after 12 months of subcutaneous implantation in rabbits; Figure 5 This is a stained photograph of a section after 12 months of subcutaneous implantation of the filler in Example 1 in rabbits; Figure 6 This is a stained photograph of the slices after 12 months of subcutaneous implantation of the filler of Example 3 in rabbits; Figure 7 This is a stained section photograph of the filler of Example 3 after subcutaneous implantation in rabbits for 12 months.

Claims

1. A method for preparing an injectable collagen filler, characterized in that: The following steps are involved: (S1) The extracted collagen solution is subjected to salting-out precipitation, and the precipitate is dialyzed and freeze-dried to obtain collagen; (S2) dissolving collagen with hydrochloric acid and then homogenizing with PBS buffer solution to prepare a non-cross-linked collagen gel; (S3) Dissolving collagen with hydrochloric acid and then homogenizing with PBS buffer solution, irradiating the obtained collagen gel by gradient irradiation cross-linking, and granulating with a mesh after irradiation to obtain a cross-linked collagen gel; the gradient irradiation cross-linking is that the irradiation intensity is gradually increased from 10-15 kgy to 25-30 kgy, and then the irradiation intensity is maintained; (S4) Mixing the non-cross-linked collagen gel obtained in step (S2), the cross-linked collagen gel obtained in step (S3), and the microspheres for injection filling, degassing and filling, and obtaining an injectable collagen filler.

2. The preparation method according to claim 1, characterized in that: In step (S1), the collagen source includes but is not limited to animal root tendon, animal skin, bovine pericardium, and arachnoid membrane.

3. The preparation method according to claim 1, characterized in that: When preparing non-cross-linked collagen in (S2), the collagen is derived from animal skin; when preparing non-cross-linked collagen in step (S3), the collagen is derived from animal Achilles tendon, preferably from bovine Achilles tendon.

4. The preparation method according to claim 1, characterized in that: The concentration of collagen in the non-cross-linked collagen gel is 20-35 mg / mL, and the concentration of collagen in the cross-linked collagen gel is 25-32 mg / mL.

5. The preparation method according to claim 1, characterized in that: The pH of the hydrochloric acid in step (S2) and step (S3) is 3-4; the pH of the PBS buffer solution is 7.2-7.

6.

6. The preparation method according to claim 1, characterized in that: In step (S2), the ratio of collagen, hydrochloric acid, and PBS buffer is 1 g: 20-30 mL: 2-3 mL.

7. The preparation method according to claim 1, characterized in that: In step (S3), the gradient irradiation time is 15-30 minutes, and then the irradiation intensity is maintained for 3-5 minutes; the intensity of the gradient irradiation gradually increases in a linear manner.

8. The preparation method according to claim 7, characterized in that: In step (S3), the granulation is performed through a mesh after irradiation, and the mesh aperture is 90-150 μm.

9. The preparation method according to claim 1, characterized in that: In step (S4), the microspheres for injection filling include PCL microspheres, PLLA microspheres or PLGA microspheres, and the particle size of the microspheres for injection filling is 30-50 μm.

10. The preparation method according to claim 1, characterized in that: In step (S4), the mass ratio of non-cross-linked collagen gel, cross-linked collagen gel, and microspheres for injection filling is 2-4:3-5:1-2.

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