Injectable poly-L-lactic acid microsphere composite gel capable of inducing rapid regeneration and instant filling of collagen and preparation method of injectable poly-L-lactic acid microsphere composite gel

Abstract

The invention discloses injectable poly-L-lactic acid microsphere composite gel capable of inducing rapid regeneration and instant filling of collagen, which is characterized in that poly-L-lactic acid is used as a raw material, and poly-L-lactic acid microspheres are prepared by adopting an emulsification method; the preparation method comprises the following steps: taking collagen and oxidized polysaccharide as raw materials, carrying out Schiff alkali cross-linking reaction on the collagen and the oxidized polysaccharide to prepare composite gel, and uniformly distributing L-polylactic acid microspheres in the composite gel. The aldehyde group on the oxidized polysaccharide and the amino group on the collagen are subjected to a Schiff base cross-linking reaction to form the injectable gel with a three-dimensional network structure. The cross-linked gel provides mechanical tension to uniformly disperse the PLLA microspheres, so that the microspheres are prevented from settling or agglomerating. The composite gel can also improve the hydrophilicity of the microspheres, so that the redissolution dispersity of the microsphere filler is better, and the microspheres settle more slowly. After being injected subcutaneously, the collagen gel can be instantly filled and induce rapid regeneration of collagen, has no chemical cross-linking agent residue, and reduces the risk of inflammation (such as granuloma) caused by the organism and the like.

Description

Technical Field

[0001] The present invention belongs to the field of medical cosmetic materials and relates to a poly (L-lactic acid) (PLLA, i.e., poly (lactic acid-co-glycolic acid)) microsphere composite gel, a preparation method, and uses thereof. Specifically, it relates to an injectable poly (L-lactic acid) microsphere composite gel that can induce rapid collagen regeneration and instant filling, a preparation method, and uses thereof in preparing subcutaneous fillers. Background Art

[0002] Facial skin collagen loss, fat atrophy, and tissue defects are significant issues affecting human well-being and health. Especially in the context of an increasingly aging population, facial tissue repair has become a major challenge in the global medical field. In recent years, demand for minimally invasive surgery has continued to increase due to its advantages, including less invasiveness, faster recovery, fewer scars, and higher patient satisfaction. Currently, minimally invasive procedures account for approximately 90% of medical aesthetic interventions.

[0003] Facial fillers are the main products for minimally invasive facial surgical treatments, and their main types include botulinum neurotoxin, collagen, hyaluronic acid, polymethyl methacrylate, hydroxyapatite, and poly-L-lactic acid microspheres. Facial fillers need to provide safe, predictable treatments with a low risk of adverse events. However, early-developed products such as collagen and hyaluronic acid have the risk of inducing potential complications, including mild edema, infection, and even blindness or stroke. In recent years, most of the products approved at home and abroad are new-generation facial fillers based on biodegradable synthetic polymers (such as poly-L-lactic acid PLLA, etc.). PLLA microspheres and their degradation products are considered to be active ingredients that stimulate collagen regeneration. They can recruit macrophages, lymphocytes, and fibroblasts at the injection site, and stimulate the regeneration of endogenous collagen and dermal fibroblasts by inducing a mild inflammatory response. The therapeutic effects of PLLA microspheres typically last for 25 months. However, as a hydrophobic, biodegradable polymer, PLLA has a slow ability to induce cell migration and a long degradation cycle. Studies have shown that the onset of effect of single PLLA microsphere facial fillers takes 3-6 months. Therefore, the collagen induction effect is limited in the initial treatment period, and the treatment period varies depending on the product. Simple polysaccharide fillers, such as hyaluronic acid, have a low molecular weight and are easily degraded by hyaluronidase or lysozyme. They are rapidly cleared through the lymphatic system and have a half-life of only a few hours in the body. Deep fillers are prone to displacement and are unable to stimulate collagen regeneration. Uncross-linked collagen materials are easily degraded by collagenase (MMP-1) in the body to form small peptide fragments that are phagocytosed and cleared by macrophages. They also have weak mechanical strength and support. Currently, there are also cross-linked polysaccharide or collagen fillers on the market, but most are chemically cross-linked with cross-linkers such as glutaraldehyde and BDDE. The cross-linker residues are prone to occur, leading to the risk of inflammation such as granulomas. Summary of the Invention

[0004] The purpose of the present invention is to develop an injectable PLLA microsphere composite gel and its use that promotes rapid collagen regeneration and instant filling, in response to the shortcomings of current subcutaneous filler products with a single PLLA as the main component and polysaccharide or collagen fillers.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] The invention relates to an injectable poly (L-lactic acid) (PLLA) microsphere composite gel that can induce rapid collagen regeneration and instant filling. The composite gel is prepared from poly (L-lactic acid) as the raw material and the PLLA microspheres are prepared by an emulsification method. The composite gel is prepared from collagen and oxidized polysaccharide as the raw materials through a Schiff base cross-linking reaction between the collagen and the oxidized polysaccharide, and the PLLA microspheres are evenly distributed in the composite gel.

[0007] Preferably, the injectable PLLA microsphere composite gel that integrates rapid collagen regeneration induction and instant filling is prepared by dissolving PLLA in dichloromethane or chloroform and preparing PLLA microspheres by an emulsification method; adding sodium periodate to a polysaccharide aqueous solution for oxidation reaction, terminating the reaction with ethylene glycol after the reaction, and dialyzing to obtain oxidized polysaccharide; mixing the oxidized polysaccharide solution and the collagen solution at a temperature of 4 to 8° C., adding PLLA microspheres, and then performing a Schiff base cross-linking reaction at a temperature of 20 to 30° C. to obtain the injectable PLLA microsphere composite gel.

[0008] Another object of the present invention is to provide a method for preparing the injectable PLLA microsphere composite gel that can induce rapid collagen regeneration and instant filling, comprising:

[0009] Step (1), using poly (L-lactic acid) (PLLA) as raw material, preparing PLLA microspheres by emulsification method;

[0010] Step (2), using polysaccharide as raw material, water as reaction solvent, and sodium periodate as oxidant, to prepare oxidized polysaccharide through oxidation reaction;

[0011] Step (3), respectively preparing a collagen solution and an oxidized polysaccharide solution;

[0012] Step (4): mixing the collagen solution and the oxidized polysaccharide solution at a temperature of 4 to 8° C., adding PLLA microspheres and mixing them evenly, and then performing a Schiff base cross-linking reaction at a temperature of 20 to 30° C. to obtain an injectable PLLA microsphere composite gel.

[0013] In step (1), PLLA microspheres are prepared by an emulsification method, and the operation is as follows: dissolving L-polylactic acid in dichloromethane or chloroform to prepare a L-polylactic acid solution; using deionized water to prepare a surfactant solution; adding the L-polylactic acid solution dropwise to the surfactant solution, after the dropwise addition is completed, fully mixing, and then stirring at a speed of 300 to 3000 rpm for 48 hours, centrifuging, collecting the precipitate, washing the precipitate with deionized water, and obtaining PLLA microspheres by freeze-drying.

[0014] The intrinsic viscosity of the L-polylactic acid is 1.0-4.0 dl / g, preferably 1.8 dl / g.

[0015] The surfactant is selected from polyvinyl alcohol, sodium lauryl sulfate, Tween, Span, sodium lauryl sulfate, cocamidopropyl betaine, cetyltrimethylammonium chloride, sodium lauryl sulfate, cocamidopropyl hydroxysulfonyl betaine, etc., preferably polyvinyl alcohol.

[0016] The viscosity of the polyvinyl alcohol is 25.0-31.0 Pa·s.

[0017] The concentration of the surfactant in the surfactant solution is 0.0005-0.05 g / mL, preferably 0.01 g / mL.

[0018] The mass ratio of the L-polylactic acid to the surfactant is 0.2:1 to 0.7:1.5, preferably 0.25:1.

[0019] The volume ratio of the dichloromethane or chloroform to deionized water is 1:5 to 1:30, preferably 1:20.

[0020] Preferably, when the L-polylactic acid solution is added dropwise, the rotation speed is controlled at 800 rpm; after the addition is completed, the rotation speed is maintained at 800 rpm and stirred for 0.5 h to ensure sufficient mixing and uniform dispersion; then, the rotation speed is increased to 1000 rpm and stirred for 48 h to prepare microspheres.

[0021] Preferably, at room temperature (25°C), the rotation speed is controlled at 800 rpm, and the L-polylactic acid solution is added dropwise to the surfactant solution while stirring; after the dropwise addition is completed, the temperature is maintained at room temperature (25°C), and the mixture is stirred at 800 rpm for 0.5 h to fully mix, and then stirred at 1000 rpm for 48 hours, centrifuged, and the precipitate is collected. The precipitate is washed with deionized water and freeze-dried to obtain microspheres.

[0022] The rotation speed of the centrifugal treatment is 3000 rpm, and the time of the centrifugal treatment is 5 to 8 minutes.

[0023] In step (2), the preparation method of the oxidized polysaccharide is as follows: dissolving the polysaccharide in deionized water to prepare a polysaccharide aqueous solution, adding sodium periodate to the polysaccharide aqueous solution, carrying out an oxidation reaction under light-proof conditions (darkness) to generate the oxidized polysaccharide, adding ethylene glycol to terminate the oxidation reaction, and dialyzing the solution in deionized water with a dialysis bag to remove by-products and unreacted oxidant, and freeze-drying to obtain the oxidized polysaccharide.

[0024] The polysaccharide is selected from one or a combination of hyaluronic acid, sodium alginate, dextran, carboxymethyl cellulose and the like.

[0025] The molecular weight of the polysaccharide is 10kDa to 10000kDa.

[0026] The concentration of the polysaccharide aqueous solution is 3 mg / mL to 30 mg / mL, preferably 10 mg / mL.

[0027] The sodium periodate is added in the form of a solution, and the concentration of the sodium periodate solution is 0.1 mg / mL to 1 mg / mL, preferably 0.5 mg / mL.

[0028] The mass ratio of the polysaccharide to sodium periodate is 1:0.5 to 1:5, preferably 1:1.

[0029] The molecular weight cut-off (MWCO) of the dialysis bag is 500Da to 20,000Da, preferably 3,500Da.

[0030] The reaction temperature is room temperature, and the reaction time is 4 to 72 hours, preferably 48 hours.

[0031] The dialysis was performed using deionized water for 3 days, with the dialysate being replaced every 2 hours on the first day and every 5 hours from the second day onwards.

[0032] In step (3), the collagen is selected from gelatin, recombinant type III collagen, recombinant bovine collagen, etc.

[0033] The preparation method of the collagen solution includes: dissolving collagen in a weakly acidic buffer solution, and after complete dissolution, adjusting the pH to 7.4 with a 0.1 mol / L sodium hydroxide solution to ensure that the collagen amino group (-NH2) is in a non-protonated state to facilitate reaction with the aldehyde group. The concentration of the collagen is 0.5 mg / mL to 20 mg / mL, preferably 10 mg / mL.

[0034] The weakly acidic buffer solution is a PBS buffer solution with a pH of 3.0.

[0035] The method for preparing the oxidized polysaccharide solution comprises: using PBS buffer (pH 7.4) to prepare the oxidized polysaccharide solution.

[0036] In step (4), the mass ratio of the oxidized polysaccharide to collagen is 1:0.5 to 1:5, preferably 1:1.

[0037] The mass ratio of the total amount of the oxidized polysaccharide and collagen (ie the amount of the composite gel) to the PLLA microspheres is 1:0.01 to 1:10, preferably 1:1.

[0038] Preferably, the oxidized polysaccharide solution and collagen solution are mixed at 4°C, and the PLLA microspheres are quickly added and mixed evenly. The mixture is then subjected to a Schiff base cross-linking reaction at 25°C to produce an injectable PLLA microsphere composite gel. The Schiff base cross-linking reaction between the oxidized polysaccharide and collagen forms a gel that not only effectively replaces conventional fillers such as carboxymethyl cellulose, polyethylene glycol, and mannitol, but also exhibits excellent microsphere dispersion, mechanical support, and bioactivity. The gel's unique mechanical tension support and effective dispersion of the PLLA microspheres prevent the PLLA microspheres from agglomerating and settling. When injected into the body, the PLLA microsphere composite gel combines mechanical support and bioactivity, instantly filling skin grooves. After degradation in the body, the collagen rapidly stimulates collagen regeneration. This gel prevents degradation and clearance of the polysaccharide and collagen alone. Furthermore, the PLLA microsphere composite gel lacks residual chemical cross-linkers (Schiff base bonds), reducing the risk of inflammation (such as granulomas) in the body.

[0039] Experimental verification shows that when the concentration of the dispersant (such as carboxymethyl cellulose) is too low, the microspheres in the traditional system are prone to sedimentation and uneven dispersion, while the composite gel of the present invention can effectively avoid this problem. This is mainly due to its unique three-dimensional network structure. The mechanical tension provided by the cross-linked gel can provide uniform support and stabilization for the microspheres, thereby ensuring the stable dispersion of the microspheres in the gel system. In addition, the microsphere composite gel of the present invention shows good performance in terms of injectability, biocompatibility and in vivo degradability, breaking through the bottleneck of "fast degradation and weak regeneration" of traditional materials, combining instant filling and long-term regeneration functions, and avoiding the toxicity risk of chemical cross-linking agents, and has broad application prospects.

[0040] Another object of the present invention is to provide use of the injectable PLLA microsphere composite gel that integrates the induction of rapid collagen regeneration and instant filling in the preparation of subcutaneous fillers.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] Compared to existing PLLA microsphere subdermal fillers, the present invention oxidizes polysaccharides with sodium periodate to form oxidized polysaccharides with aldehyde groups. The aldehyde groups on the oxidized polysaccharide react with amino groups on collagen through a Schiff base cross-linking reaction to form an injectable gel with a three-dimensional network structure. The cross-linked gel provides mechanical tension to evenly disperse the PLLA microspheres, preventing them from settling or agglomerating, thus forming a novel PLLA microsphere composite gel subdermal filler. The composite gel also improves the hydrophilicity of the microspheres, resulting in better redispersibility and dispersion of the microsphere filler, slower microsphere sedimentation, and greater ease of use. Furthermore, the composite gel exhibits excellent performance in terms of injectability, biocompatibility, and in vivo degradability, promising broad application prospects.

[0043] In terms of the injection mechanism, the PLLA microsphere composite gel of the present invention can quickly fill tissue wrinkles or grooves. After injection, the collagen in the composite gel can quickly induce collagen regeneration in the body, demonstrating excellent tissue repair capabilities.

[0044] Compared to existing preparation processes, the PLLA microsphere composite gel subcutaneous filler, formed through Schiff base crosslinking, is simple to operate and easy to scale up, making it ideally suited for factory production. This not only simplifies the process but also improves production efficiency and reduces production costs, providing strong support for industrialized production.

[0045] Compared to existing PLLA microsphere fillers using carboxymethyl cellulose, this invention utilizes a highly biocompatible oxidized polysaccharide covalently cross-linked with collagen to form a gel-based PLLA microsphere system. This overcomes the bottleneck of traditional fillers' rapid degradation and weak regeneration, combining immediate filling with long-term regeneration while avoiding the toxicity risks of chemical cross-linkers. This composite gel possesses high clinical translational value and avoids the biocompatibility and osmotic pressure issues associated with carboxymethyl cellulose. Furthermore, this composite gel exhibits superior mechanical properties and stability, further enhancing its advantages in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a scanning electron microscope image of PLLA microspheres.

[0047] Figure 2 This is the collagen production of injectable PLLA microsphere composite gel subdermal filler implanted in vivo for 2 weeks. DETAILED DESCRIPTION

[0048] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0049] Example 1

[0050] The preparation method of PLLA microspheres is as follows:

[0051] 15g of poly (L-lactic acid) (PLLA) with an intrinsic viscosity of 1.8 dl / g was weighed and added to 300mL of dichloromethane and dissolved thoroughly to obtain a 0.05g / mL PLLA solution. 60g of poly (vinyl alcohol) (PVA) with a viscosity of 25-31 mPa·s was weighed and added to 6000mL of deionized water and dissolved completely to obtain a 0.01g / mL PVA solution. At room temperature (25°C), the PLLA solution was slowly added dropwise to the PVA solution under magnetic stirring at 600rpm. After the addition was complete, stirring was continued at the same speed for 0.5 hours to ensure that the mixture was fully dispersed and evenly mixed. Subsequently, the stirring mode was switched to mechanical stirring at 1000rpm at room temperature (25°C) and continued for 48 hours to promote microsphere formation.

[0052] The reaction solution was centrifuged at 3000 rpm for 5 minutes to obtain a precipitate. The precipitate was dispersed with deionized water and centrifuged again at 3000 rpm for 5 minutes. The washing and centrifugation operations were repeated eight times to completely remove the polyvinyl alcohol. The precipitate was collected by centrifugation and freeze-dried to obtain PLLA microspheres.

[0053] Weigh 10 mg of PLLA microsphere powder and dissolve it in 1 mL of normal saline. After 1 hour, observe the microsphere morphology under an inverted microscope. The results show that the PLLA microspheres are uniformly spherical, ranging in size from 20 to 100 μm.

[0054] Take 16g of hyaluronic acid with a molecular weight of about 100kDa and place it in a beaker. Add 1600mL of deionized water. After it is fully dissolved, slowly add 160mL of sodium periodate solution with a concentration of 2.67% (w / v), and gently stir to mix. Place the beaker in a dark environment and continue the reaction for 48 hours at room temperature (25°C) to ensure the full oxidation of the polysaccharide. After the reaction is completed, 80mL of ethylene glycol is quickly added to effectively terminate the oxidation reaction. Collect the reaction mixture into a dialysis bag (MWCO=3500Da) and dialyze with deionized water for 3 days. During the first day, the dialysate is replaced every two hours, and the dialysate is replaced every 5 hours from the second day to thoroughly remove small molecule impurities and unreacted reagents. After the dialysis is completed, oxidized hyaluronic acid is obtained by freeze drying.

[0055] Take type I collagen with a molecular weight of approximately 50-100 kDa and pre-cooled PBS buffer (pH 3.0) to 4°C. Under stirring conditions, add type I collagen to the PBS buffer at a stirring speed of approximately 800 rpm to ensure that the collagen is evenly and fully dispersed in the PBS buffer to avoid local excessive concentration or agglomeration, thereby maintaining the integrity of the collagen molecular structure and preventing excessive shear force from causing collagen denaturation and inactivation. Finally, a collagen solution with a concentration of 10 mg / mL is obtained. The pH is adjusted to 7.4 with 0.1 mol / L sodium hydroxide solution under stirring and stored in a 4°C refrigerator for use.

[0056] Oxidized hyaluronic acid was dissolved in PBS buffer (pH 7.4) to obtain an oxidized hyaluronic acid solution with a concentration of 10 mg / mL, which was stored in a refrigerator at 4° C. until use.

[0057] Take 1 mL of collagen solution and 1 mL of oxidized hyaluronic acid solution, mix them at a temperature of 4°C, quickly add 20 mg of PLLA microsphere powder, vortex to mix evenly to promote sufficient Schiff base cross-linking reaction between the subsequent collagen and oxidized polysaccharide, then heat to 25°C, carry out Schiff base cross-linking reaction under stirring, so that the PLLA microspheres are evenly distributed in the composite gel, and freeze-dry to obtain a PLLA microsphere composite gel.

[0058] The PLLA microsphere composite gel prepared in this example was dissolved in 2 mL of deionized water to obtain a filler. 200 μL of the filler was injected into the subcutaneous tissue of the back of healthy SD rats (190 g to 200 g). Two weeks later, the subcutaneous tissue of the rat back was dissected and embedded. The microspheres were observed and photographed under a light microscope. It was found that the microspheres were surrounded by a large amount of Col. III collagen, indicating that they have the ability to promote the rapid regeneration of type III collagen ( Figure 2 This result strongly indicates that the PLLA microsphere composite gel of the present invention can quickly and effectively stimulate the body's collagen regeneration mechanism, thereby significantly accelerating the tissue repair and regeneration process.

Claims

1. An injectable poly (L-lactic acid) microsphere composite gel that can induce rapid collagen regeneration and instant filling, characterized by: It uses L-lactic acid as raw material and adopts emulsification method to prepare L-lactic acid microspheres; uses collagen and oxidized polysaccharide as raw materials, and uses Schiff base cross-linking reaction of collagen and oxidized polysaccharide to prepare composite gel, and the L-lactic acid microspheres are evenly distributed in the composite gel.

2. The injectable poly (L-lactic acid) microsphere composite gel capable of inducing rapid collagen regeneration and instant filling according to claim 1, characterized in that: The method comprises dissolving L-polylactic acid in dichloromethane or chloroform and preparing L-polylactic acid microspheres by an emulsification method; adding sodium periodate to a polysaccharide aqueous solution for oxidation reaction, terminating the reaction with ethylene glycol after the reaction, and dialyzing to obtain oxidized polysaccharide; stirring and mixing the oxidized polysaccharide solution and collagen solution at a temperature of 4 to 8°C, adding L-polylactic acid microspheres, and then performing a Schiff base cross-linking reaction at a temperature of 20 to 30°C to obtain an injectable L-polylactic acid microsphere composite gel.

3. A method for preparing the injectable poly (L-lactic acid) microsphere composite gel capable of inducing rapid collagen regeneration and instant filling, characterized by: include: Step (1), using L-polylactic acid as raw material, preparing L-polylactic acid microspheres by emulsification method; Step (2), using polysaccharide as raw material, water as reaction solvent, and sodium periodate as oxidant, to prepare oxidized polysaccharide through oxidation reaction; Step (3), respectively preparing a collagen solution and an oxidized polysaccharide solution; Step (4): mixing the collagen solution and the oxidized polysaccharide solution at a temperature of 4 to 8° C., adding the L-polylactic acid microspheres and mixing them evenly, and then performing a Schiff base cross-linking reaction at a temperature of 20 to 30° C. to obtain an injectable L-polylactic acid microsphere composite gel.

4. The method for preparing the injectable poly (L-lactic acid) microsphere composite gel capable of inducing rapid collagen regeneration and instant filling according to claim 3, characterized in that: In step (1), L-polylactic acid microspheres are prepared by an emulsification method, including: dissolving L-polylactic acid in dichloromethane or chloroform to prepare a L-polylactic acid solution; using deionized water to prepare a surfactant solution; adding the L-polylactic acid solution dropwise to the surfactant solution, after the dropwise addition is completed, fully mixing, then stirring at a speed of 300 to 3000 rpm for 48 hours, centrifuging, collecting the precipitate, washing with deionized water, and freeze-drying to obtain L-polylactic acid microspheres.

5. The method for preparing the injectable poly (L-lactic acid) microsphere composite gel capable of inducing rapid collagen regeneration and instant filling according to claim 3 or 4, characterized in that: In step (1), the intrinsic viscosity of the left-handed polylactic acid is 1.0 to 4.0 dl / g, preferably 1.8 dl / g; the surfactant is selected from polyvinyl alcohol, sodium lauryl sulfate, Tween, Span, sodium lauryl sulfate, cocamidopropyl betaine, hexadecyl trimethyl ammonium chloride, sodium lauryl sulfate, cocamidopropyl hydroxysulfonyl betaine, etc., preferably polyvinyl alcohol; the concentration of the surfactant in the surfactant solution is 0.0005 to 0.05 g / mL, preferably 0.01 g / mL; the mass ratio of the left-handed polylactic acid and the surfactant is 0.2:1 to 0.7:1.5, preferably 0.25:1; the volume ratio of the dichloromethane or chloroform to deionized water is 1:5 to 1:30, preferably 1:

20.

6. The method for preparing the injectable poly (L-lactic acid) microsphere composite gel capable of inducing rapid collagen regeneration and instant filling according to claim 3, characterized in that: In step (2), the preparation method of the oxidized polysaccharide is as follows: dissolving the polysaccharide in deionized water to prepare a polysaccharide aqueous solution, adding sodium periodate to the polysaccharide aqueous solution, carrying out an oxidation reaction under light-proof conditions to generate the oxidized polysaccharide, adding ethylene glycol to terminate the oxidation reaction, and dialyzing the solution in deionized water with a dialysis bag to remove by-products and unreacted oxidant, and freeze-drying to obtain the oxidized polysaccharide.

7. The method for preparing the injectable poly (L-lactic acid) microsphere composite gel capable of inducing rapid collagen regeneration and instant filling according to claim 3 or 5, characterized in that: In step (2), the polysaccharide is selected from one or a combination of hyaluronic acid, sodium alginate, dextran, and carboxymethyl cellulose; the molecular weight of the polysaccharide is 10 kDa to 10,000 kDa; the mass ratio of the polysaccharide to sodium periodate is 1:0.5 to 1:5, preferably 1:1; the molecular weight cut-off of the dialysis bag is 500 Da to 20,000 Da, preferably 3,500 Da; In step (3), the collagen is selected from gelatin, recombinant type III collagen, and recombinant bovine collagen; The preparation method of the collagen solution comprises: dissolving collagen in a weakly acidic buffer solution, and adjusting the pH to 7.4 after complete dissolution, wherein the concentration of the collagen is 0.5 mg / mL to 20 mg / mL, preferably 10 mg / mL; The method for preparing the oxidized polysaccharide solution comprises: using pH 7.4 PBS buffer to prepare the oxidized polysaccharide solution.

8. The method for preparing the injectable poly (L-lactic acid) microsphere composite gel capable of inducing rapid collagen regeneration and instant filling according to claim 3, characterized in that: In step (4), the mass ratio of the oxidized polysaccharide to collagen is 1:0.5 to 1:5; the mass ratio of the total amount of the oxidized polysaccharide and collagen to the poly (L-lactic acid) microspheres is 1:0.01 to 1:

10.

9. The method for preparing the injectable poly (L-lactic acid) microsphere composite gel capable of inducing rapid collagen regeneration and instant filling according to claim 8, characterized in that: In step (4), the mass ratio of the oxidized polysaccharide to the collagen is 1:1; the mass ratio of the total amount of the oxidized polysaccharide and collagen to the poly (L-lactic acid) microspheres is 1:

1.

10. Use of the injectable poly (L-lactic acid) microsphere composite gel capable of inducing rapid collagen regeneration and instant filling as claimed in claim 1 in the preparation of a subcutaneous filler.

Images