Injection filler capable of durably suspending to stimulate collagen regeneration and preparation method thereof

Abstract

The invention provides an injection filler capable of persistently suspending and stimulating collagen regeneration as well as a preparation method and application of the injection filler, and belongs to the technical fields of medicines and cosmetic filling materials. The composite microsphere is composed of porous composite microspheres and hydrogel, the porous composite microsphere material is a polylactic acid microsphere and hydroxyapatite nano microsphere composite porous microsphere; the hydrogel comprises a matrix material, water, an isotonic agent, a pH regulator and an analgesic. The prepared injection filler porous composite microspheres capable of being persistently suspended to stimulate collagen regeneration have adjustable density, are not easy to aggregate and condense, cannot cause nodules after being injected into a human body, and can stimulate collagen regeneration.

Description

Technical Field

[0001] This invention belongs to the fields of medicine and cosmetic filler materials, specifically relating to an injectable filler that can persistently suspend and stimulate collagen regeneration, its preparation method, and its application. Background Technology

[0002] Currently, there are many injectable filler products on the market, which can be divided into two main categories based on their mechanism of action: The first category provides a filling effect through volume, such as cross-linked hyaluronic acid and collagen fillers. This type of filler is not permanent and cannot provide long-term filling effects, requiring multiple injections. Additionally, swelling may occur shortly after injection, and the water absorption of hyaluronic acid itself can also cause localized edema, especially in areas like the lips. Furthermore, overfilling can lead to a "puffy face" appearance. The second category of fillers stimulates the body's own collagen regeneration to achieve a filling effect, including high-molecular-weight synthetic polymers such as polycaprolactone microspheres, polylactic acid microspheres, and hydroxyapatite. This type of filler primarily stimulates collagen regeneration through degradation products, providing long-term filling effects. However, it works slowly after injection and is prone to aggregation, leading to facial nodules.

[0003] Polylactic acid (PLA) is a biocompatible and biodegradable polymer material. It is not only safe and harmless to the human body, but its degradation products can also safely participate in the body's metabolic processes. However, PLA is a highly hydrophobic material with no cell-specific sites on its surface, and its degradation products are acidic. These characteristics can easily cause aseptic inflammatory reactions at the implantation site, adversely affecting cell adhesion and growth. Furthermore, because it is mostly in solid spheres, the product degrades slowly, resulting in a slow onset of action after injection, impacting the user experience and failing to produce an immediate filling effect.

[0004] Hydroxyapatite (HAP) is a major inorganic component of human bones and teeth, and has attracted much attention due to its excellent biocompatibility and bioactivity. Compared with polylactic acid (PLA), hydroxyapatite has better mechanical properties, a longer degradation cycle, and stronger thermal stability and hydrophilicity. Therefore, combining PLA with hydroxyapatite not only improves the mechanical properties of PLA and extends its degradation cycle, but also facilitates cell and protein adsorption, thereby enhancing the bioactivity of the composite material. Furthermore, because hydroxyapatite is weakly alkaline, it can neutralize the acidic substances produced during PLA degradation, thus mitigating the resulting aseptic inflammatory response.

[0005] Currently, there are two main methods for preparing polylactic acid (PLA) / hydroxyapatite (hydroxyapatite) composite microspheres: 1) Dispersing hydroxyapatite ultrasonically in a prepared PLA solution to form a suspension, then adding the suspension to a polyvinyl alcohol solution, and preparing PLA / hydroxyapatite composite microspheres through emulsification solvent evaporation; 2) Preparing PLA microspheres containing calcium carbonate using microfluidic technology, then immersing the microspheres in a phosphate solution, and obtaining PLA / hydroxyapatite composite microspheres through a mineralization reaction. The PLA / hydroxyapatite composite microspheres prepared by the above two methods are either solid spheres or have very small surface pores, which are not conducive to cell adhesion, growth, and proliferation. This technology provides a porous PLA / hydroxyapatite composite microsphere with controllable pore size, and the mineralized hydroxyapatite coating exists not only on the surface of the microsphere but also inside, which is more conducive to cell adhesion, growth, and proliferation compared to traditionally prepared composite microspheres.

[0006] CN111286074A discloses a method for preparing polylactic acid / nano-hydroxyapatite composite microspheres. The method involves adding nano-hydroxyapatite to a prepared polylactic acid solution, dispersing it by ultrasound to form a suspension, then adding the suspension to a polyvinyl alcohol solution, stirring, allowing it to stand, filtering, washing, and freeze-drying to obtain the polylactic acid / nano-hydroxyapatite composite microspheres. The microspheres prepared by this method are solid spheres without a porous structure, which is detrimental to cell attachment and proliferation.

[0007] CN107519536A discloses a method for preparing porous polylactic acid / hydroxyapatite composite microspheres. This method also involves adding hydroxyapatite to a prepared polylactic acid solution, dispersing it by ultrasound to form a suspension, then adding the suspension to a polyvinyl alcohol solution, stirring, allowing it to stand, separating the solid and liquid phases, washing, and drying to obtain polylactic acid / hydroxyapatite composite microspheres with a porous surface structure. Unlike the previous method, this method uses hydroxyapatite as a pore-forming agent, controlling the pore size of the microspheres by incorporating hydroxyapatite with different crystal sizes. Although the microspheres prepared by this method have a porous structure, the pore size is relatively small, which is not conducive to cell entry and exit. Furthermore, the pore-forming process using hydroxyapatite requires high precision in the physical dimensions of the hydroxyapatite material.

[0008] CN109749119A discloses a method for preparing polylactic acid / hydroxyapatite micron- to nano-level hierarchical composite microspheres. This method involves dissolving polylactic acid and calcium carbonate together in dichloromethane, then using microfluidic technology to dropwise add the solution into a polyvinyl alcohol solution under stirring conditions to obtain polylactic acid microspheres containing calcium carbonate. These microspheres are then immersed in a K₂HPO₄ solution for a mineralization reaction. After harvesting, the microspheres are washed and dried to obtain the polylactic acid / hydroxyapatite micron- to nano-level hierarchical composite microsphere material. However, the microspheres prepared by this method are solid spheres without a porous structure, and the microfluidic technology required is sophisticated, resulting in high equipment costs and hindering large-scale production.

[0009] This invention aims to solve one of the technical problems existing in the prior art. To this end, this invention proposes an injectable filler that can sustainably suspend and stimulate collagen regeneration. The microspheres are characterized by a porous structure, which facilitates cell entry, adhesion, and proliferation. Furthermore, the microspheres have controllable pore size, uniform particle size, and high sphericity, making them suitable for various applications such as cosmetic filling and bone repair. In addition, this invention proposes a method for preparing the injectable filler that can sustainably suspend and stimulate collagen regeneration. This method is not simply about adding hydroxyapatite to a polylactic acid solution and then preparing polylactic acid / hydroxyapatite composite microspheres through an emulsification solvent evaporation method. Instead, it first prepares porous polylactic acid microspheres using a double emulsification solvent evaporation method, then adds the porous polylactic acid microspheres to a mineralization solution, and under certain reaction conditions, mineralizes a layer of hydroxyapatite microspheres on the porous polylactic acid microspheres. Compared with traditional methods, the polylactic acid / hydroxyapatite composite microspheres prepared by this method have a porous structure with controllable particle size and pore size. The prepared microspheres have better sphericity and are easier to mineralize. The mineralization process combines polylactic acid microspheres with hydroxyapatite microspheres, which not only improves the hydrophilicity of polylactic acid microspheres, thus facilitating cell and protein adsorption and advancing the time for polylactic acid microspheres to stimulate collagen regeneration, but also allows hydroxyapatite microspheres to adjust the density of polylactic acid microspheres, preventing microsphere aggregation and the occurrence of facial nodules. In addition, the weakly alkaline properties of hydroxyapatite can partially neutralize the acid effect produced by polylactic acid materials during degradation, reducing local inflammatory reactions caused by acidity around the implanted material. Summary of the Invention

[0010] Purpose of the invention: To address the problems existing in the prior art, this invention provides an injectable filler that can stimulate collagen regeneration and remodeling. This invention adds specific porous composite microspheres into hydrogel to form a special spatial structure, which can produce an immediate filling effect after subcutaneous injection. As the hydrogel is absorbed, the porous composite microspheres are gradually exposed as foreign body stimulation, causing only short-term mild inflammation in the early stage and reducing the risk of granulomas. It significantly increases the total amount of collagen and remodels the ratio of stimulated collagen type I and type III, making it closer to normal skin.

[0011] The present invention also provides a method for preparing and applying the injectable filler that can stimulate collagen regeneration and remodeling.

[0012] This technical solution: In order to achieve the above objective, an injectable filler that can persistently suspend and stimulate collagen regeneration is provided, characterized in that the filler is mainly composed of porous composite microspheres and hydrogel; the porous composite microsphere material is a composite microsphere of porous polylactic acid microspheres and hydroxyapatite nanospheres, and the hydrogel includes matrix material, water, isotonic agent, pH adjuster, and analgesic.

[0013] The porous composite microspheres contain polylactic acid and hydroxyapatite in a mass ratio of 3:1 to 5:1, and the porous composite microspheres contain a matrix in a mass ratio of 1:30 to 1:10.

[0014] The porous composite microspheres are spherical or near-spherical, with a particle size of 10–120 μm and a pore size of 0.001–10 μm; preferably, the particle size of the porous composite microspheres is 40–80 μm and the pore size is 2–10 μm. Figure 1 The pore structure of the porous composite microspheres can be seen from the data.

[0015] The porous polylactic acid microspheres have a particle size of 40–80 μm, and the pore size is preferably 2–10 μm. Figure 2 The pore structure of the porous polylactic acid microspheres can be seen from the image.

[0016] The hydroxyapatite nanospheres have a particle size of 0.1–5 μm and a pore size of 1–100 nm. Figure 1 As can be seen, the hydroxyapatite microspheres have a nanostructure.

[0017] The hydrogel includes a matrix material, water, an isotonic agent, a pH adjuster, and an analgesic.

[0018] The matrix material is one or both of silk fibroin and collagen.

[0019] The preparation method of the injectable filler that can persistently suspend and stimulate collagen regeneration includes the following steps:

[0020] Methods for preparing porous polylactic acid microspheres include emulsification.

[0021] Methods for preparing porous composite microspheres include mineralization.

[0022] The hydrogel was prepared by chemical cross-linking.

[0023] The porous composite microspheres are added to the hydrogel and stirred thoroughly to obtain the final product.

[0024] The method for preparing the injectable filler that can persistently suspend and stimulate collagen regeneration includes the following steps:

[0025] (1) Preparation of porous polylactic acid microspheres:

[0026] Polylactic acid (PLA) is dissolved in an organic solvent to form an oil phase; gelatin or ammonium bicarbonate is completely dissolved in water to form an inner aqueous phase; polyvinyl alcohol (PVA) is dissolved in water to form an outer aqueous phase. The inner aqueous phase is slowly added to the oil phase and emulsified at a rate of 3000–8000 rpm / min for 1–10 min to form a primary emulsion; the outer aqueous phase is slowly added to the primary emulsion and emulsified at a rate of 1500–4000 rpm / min for 3–40 min; the mixture is then vacuum stirred at 100–600 rpm / min for 6–24 h to solidify; after the organic solvent in the solution has evaporated, the mixture is washed, dried, and passed through a sieve to obtain porous PLA microspheres.

[0027] (2) Preparation of porous composite microspheres:

[0028] Porous polylactic acid microspheres were immersed in a solution containing 0.05–0.2 M calcium chloride and 0.01–0.05 M magnesium chloride for 1–10 minutes. After immersion, they were removed and rinsed for 1–10 minutes. Then, they were immersed in a solution containing 0.05–0.2 M disodium hydrogen phosphate and 0.01–0.05 M sodium hydrochloride for 1–10 minutes. After immersion, they were removed and rinsed for 1–10 minutes. This alternating immersion process was repeated 2–4 times to complete the pre-mineralization.

[0029] Sodium chloride, dipotassium hydrogen phosphate, magnesium chloride, 4-hydroxyethylpiperazine ethanesulfonic acid, calcium chloride, and sodium bicarbonate were mixed in a weight ratio of 22:1:1:12:1:2 to prepare a mixed solution. The pH of the mixed solution was adjusted to 6.4-7.2 and stirred until clear to form a mineralized solution.

[0030] The pre-mineralized porous polylactic acid microspheres were placed in a mineralization solution and kept at 37°C for 1–2 days. After the solution was removed, the microspheres were filtered, washed, and dried to obtain porous composite microspheres.

[0031] (3) Preparation of hydrogel: Weigh one or two of collagen and silk fibroin, mix with water and isotonic agent, and the concentration is 0.5% to 2%.

[0032] (4) Mixing: Add the porous composite microspheres to the hydrogel, stir and mix thoroughly, then immediately dispense, freeze dry and sterilize by irradiation to obtain the filler.

[0033] Among them, the injectable filler that can persistently suspend and stimulate collagen regeneration has applications in medical aesthetics, plastic surgery, filling, and tissue defect repair.

[0034] The present invention has the following beneficial effects:

[0035] (1) In this invention, hydroxyapatite nanospheres are added to the filler formulation. After being implanted into the human body, they hydrolyze to become slightly alkaline, which reduces the occurrence of inflammation and swelling.

[0036] (2) The addition of hydroxyapatite nanospheres in this invention can adjust the density of polylactic acid microspheres, which can better disperse L-type polylactic acid microparticles, reduce their aggregation, and thus reduce the occurrence of subcutaneous nodules.

[0037] (3) The invention incorporates composite microspheres into the filler formulation. These microspheres can adsorb tissue fluid and allow fibroblasts to adhere, thus promoting collagen regeneration and achieving a faster filling effect. Attached Figure Description

[0038] Figure 1 The image shows a scanning electron microscope (SEM) image of the composite porous microsphere sample.

[0039] Figure 2 This is a scanning electron microscope image of a porous polylactic acid microsphere sample.

[0040] Figure 3 The diagram is for Experiment 1.

[0041] Figure 4 The diagram is for Experiment 3. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention.

[0043] Example 1

[0044] The filler formulation (wt%, the same applies to the following examples) is: 7% porous composite microspheres and 93% matrix material. The intrinsic viscosity of polylactic acid is 1.53 dL / g.

[0045] The preparation method of the filler is as follows:

[0046] (1) Preparation of porous polylactic acid microspheres:

[0047] A 4% (w / w) polylactic acid (PLA) mixed with dichloromethane solution was prepared. After complete dissolution by continuous stirring at room temperature for 12 hours, the solution was left to stand, forming the oil phase. A 3% ammonium bicarbonate aqueous solution was prepared to form the inner aqueous phase. Polyvinyl alcohol (PVA) was dissolved in water at a concentration of 2% to form the outer aqueous phase. The inner aqueous phase was slowly added to the oil phase, and emulsification was carried out at a rate of 5000 rpm / min for 2 minutes to form a primary emulsion. The outer aqueous phase was then slowly added to the primary emulsion, and emulsification was carried out at a rate of 3000 rpm / min for 15 minutes. The mixture was stirred at 200 rpm / min and solidified for 16 hours. After the organic solvent in the solution evaporated, the mixture was washed, dried, and passed through a sieve to obtain porous PLA microspheres.

[0048] (2) Preparation of porous composite microspheres:

[0049] Porous polylactic acid microspheres were immersed in a solution containing 0.05 M calcium chloride and 0.02 M magnesium chloride for 5 minutes, then removed and rinsed for 4 minutes. The microspheres were then immersed in a solution containing 0.05 M disodium hydrogen phosphate and 0.01 M sodium hydrochloride for 5 minutes, and then removed and rinsed for 5 minutes. This alternating immersion process was repeated twice to complete the pre-mineralization.

[0050] Sodium chloride, dipotassium hydrogen phosphate, magnesium chloride, 4-hydroxyethylpiperazine ethanesulfonic acid, calcium chloride, and sodium bicarbonate were mixed in a weight ratio of 22:1:1:12:1:2 to prepare a mixed solution. The pH of the mixed solution was adjusted to 7.2 and stirred until clear to form a mineralized solution.

[0051] The pre-mineralized porous polylactic acid microspheres were placed in a mineralization solution and kept at 37°C for 1 day. After the solution was removed, the microspheres were filtered, washed, and dried to obtain porous composite microspheres.

[0052] (3) Preparation of hydrogel: Weigh 0.5g of silk fibroin and mix it with 50ml of phosphate solution to dissolve it.

[0053] (4) Preparation of filler: Add 7g of composite microspheres to 93g of hydrogel, stir at 500r / min for 20-30min, then immediately dispense, freeze dry and sterilize by irradiation to obtain filler.

[0054] Example 2

[0055] The filler formulation (wt%, the same applies to the following examples) is: 10% porous composite microspheres and 90% hydrogel. The intrinsic viscosity of polylactic acid is 1.53 dL / g.

[0056] The preparation method of the filler is as follows:

[0057] (1) Preparation of porous polylactic acid microspheres:

[0058] A 5% (w / w) polylactic acid (PLA) mixed with dichloromethane solution was prepared and continuously stirred at room temperature for 12 hours until completely dissolved, forming the oil phase. A 5% gelatin aqueous solution was prepared to form the inner aqueous phase; polyvinyl alcohol was dissolved in water at a concentration of 1% to form the outer aqueous phase. The inner aqueous phase was slowly added to the oil phase and emulsified at a rate of 6000 rpm / min for 3 minutes to form the initial solution; the outer aqueous phase was slowly added to the initial solution and emulsified at a rate of 3000 rpm / min for 6 minutes; the mixture was stirred at 200 rpm / min and cured for 24 hours; after the organic solvent in the solution evaporated, the mixture was washed, freeze-dried, and passed through a sieve to obtain porous PLA microspheres.

[0059] (2) Preparation of porous composite microspheres:

[0060] Porous polylactic acid microspheres were immersed in a solution containing 0.2 M calcium chloride and 0.05 M magnesium chloride for 10 minutes, then removed and rinsed for 10 minutes. The microspheres were then immersed in a solution containing 0.2 M disodium hydrogen phosphate and 0.05 M sodium hydrochloride for 10 minutes, and then removed and rinsed for 10 minutes. This alternating immersion process was repeated twice to complete the pre-mineralization.

[0061] Sodium chloride, dipotassium hydrogen phosphate, magnesium chloride, 4-hydroxyethylpiperazine ethanesulfonic acid, calcium chloride, and sodium bicarbonate were mixed in a weight ratio of 22:1:1:12:1:2 to prepare a mixed solution. The pH of the mixed solution was adjusted to 6.4 and stirred until clear to form a mineralized solution.

[0062] The pre-mineralized porous polylactic acid microspheres were placed in a mineralization solution and kept at 37°C for 2 days. After the solution was removed, the microspheres were filtered, washed, and dried to obtain porous composite microspheres.

[0063] (3) Preparation of hydrogel: Weigh 1g of collagen and dissolve it in 50mL of 0.1mol / L phosphate buffer.

[0064] (4) Preparation of filler: Add 10g of composite microspheres to 90g of hydrogel, stir at 500r / min, and rotate in both directions for 30s for a total of 20-30min. Then immediately dispense, freeze dry and sterilize by irradiation to obtain the filler.

[0065] Comparative Example 1

[0066] The filler formulation (wt%, the same applies to the following examples) is: 7% porous composite microspheres and 93% matrix material. The intrinsic viscosity of polylactic acid is 1.53 dL / g.

[0067] The preparation method of the filler is as follows:

[0068] (1) Preparation of porous polylactic acid microspheres:

[0069] Polylactic acid (PLA) was dissolved in 1,4-dioxane to prepare a 1.5% homogeneous solution. A 1.2% (w / w) aqueous solution of ethylenediamine was added, and the solution was subjected to ammonolysis at 60°C for 60 min to obtain an aminated PLA solution. Under rapid stirring, 30 g of glycerol was added dropwise as a dispersant to the aminated PLA solution, and the mixture was stirred to form a stable emulsion. The emulsion was frozen at -15°C for 4 h to solidify the PLA droplets into microspheres. After filtration, washing, and freeze-drying, aminated PLA microspheres with a nanofiber structure were obtained.

[0070] (2) Preparation of porous composite microspheres:

[0071] Microspheres were immersed in a 1M calcium nitrate solution and placed in a 37℃ constant temperature water bath shaker for 4 days for adsorption. The microspheres with adsorbed calcium ions were then separated by filtration. The calcium-adsorbed microspheres were then immersed in a 0.8M dipotassium hydrogen phosphate solution, maintaining a Ca / P molar ratio of 1.67, for 7 days. After filtration, washing, and freeze-drying, hydroxyapatite / modified polylactic acid composite microspheres were obtained.

[0072] (3) Preparation of hydrogel: Weigh 0.5g of silk fibroin and mix it with 50ml of phosphate solution to dissolve it.

[0073] (4) Preparation of filler: Add 7g of composite microspheres to 93g of hydrogel, stir at 500r / min for 20-30min, then immediately dispense, freeze dry and irradiate sterilize to obtain filler.

[0074] Comparative Example 2

[0075] The filler formulation (wt%, the same applies to the examples below) is: 10% porous polylactic acid microspheres and 90% hydrogel. The intrinsic viscosity of polylactic acid is 1.53 dL / g.

[0076] (1) Preparation method of porous polylactic acid microspheres

[0077] A 4% (w / w) polylactic acid (PLA) mixed with dichloromethane solution was prepared. After complete dissolution by continuous stirring at room temperature for 12 hours, the solution was left to stand, forming the oil phase. A 3% ammonium bicarbonate aqueous solution was prepared to form the inner aqueous phase. Polyvinyl alcohol (PVA) was dissolved in water at a concentration of 2% to form the outer aqueous phase. The inner aqueous phase was slowly added to the oil phase, and emulsification was carried out at a rate of 5000 rpm / min for 2 minutes to form a primary emulsion. The outer aqueous phase was then slowly added to the primary emulsion, and emulsification was carried out at a rate of 3000 rpm / min for 15 minutes. The mixture was stirred at 200 rpm / min and solidified for 16 hours. After the organic solvent in the solution evaporated, the mixture was washed, dried, and passed through a sieve to obtain porous PLA microspheres.

[0078] (2) Preparation of hydrogel: Weigh 1g of collagen and dissolve it in 50mL of 0.1mol / L phosphate buffer.

[0079] (3) Preparation of filler: Add 10g of porous polylactic acid microspheres to 90g of hydrogel, stir at 500r / min, and rotate in both directions for 30s for a total of 20-30min. Then immediately dispense, freeze dry and sterilize by irradiation to obtain the filler.

[0080] Experiment 1: Evaluation of the suspension performance of the filler

[0081] Take 5 mL of water for injection and add it to 5 mL of the filler material from the examples and comparative examples, respectively. Mix well with a rotary mixer and determine the suspension properties of different samples. The suspension standard is: the suspension is homogeneous after 7 days of mixing, with no floating particles or sediment.

[0082] The suspension properties of the fillers in each embodiment and comparative example are as follows: Figure 3 As shown, a comparison between Examples 1 and 2 and Comparative Examples 1 and 2 reveals that the composite porous microspheres have a certain effect on improving suspension performance. A comparison between Example 1 and Comparative Example 1 and between Example 2 and Comparative Example 2 shows that the preparation method of the composite porous microspheres has a significant impact on the suspension properties of the porous polylactic acid microspheres, which reduces the occurrence of adverse reactions such as subcutaneous nodules after the filler is implanted subcutaneously.

[0083] Experiment 2: Evaluation of local injection reaction

[0084] SPF-grade Wistar rats were selected for the experiment, with an equal number of males and females, weighing approximately 180-230g.

[0085] Three rats were taken, and the backs of each rat were marked with four sites. Each group was injected with the solution described in the example.

[0086] 1. Fillers of Example 2, Comparative Example 1 and Comparative Example 2.

[0087] The injection method is as follows: Take 0.3 ml of the mixed filler solution from Experiment 1 and implant it subcutaneously into the back of the rat. Observe whether there is redness and swelling after 7 days and whether there are nodules or other adverse reactions after 4 weeks.

[0088] The injection reactions of the fillers in each embodiment and comparative example are shown in the table below:

[0089] Filler No. Local Injection Reaction

[0090] Example 1: No adverse reactions such as redness, swelling, or subcutaneous nodules occurred after injection.

[0091] Example 2: No adverse reactions such as redness, swelling, or subcutaneous nodules occurred after injection.

[0092] Comparative Example 1 showed a slight redness and swelling, and severe subcutaneous nodule reaction after injection.

[0093] Comparative Example 2 showed minor redness, swelling, and subcutaneous nodule reactions after injection.

[0094] Experimental results showed that the addition of composite porous microspheres to the filler resulted in no adverse reactions such as redness, swelling, or subcutaneous nodules one week after injection, indicating minimal skin irritation and suitability for soft tissue filling. The same method was used to evaluate the local injection reaction of a comparative filler product, and the results showed that adverse reactions such as redness, swelling, and subcutaneous nodules occurred one week after injection.

[0095] Experiment 3: Evaluation of Stimulation of Collagen Regeneration

[0096] SPF-grade Wistar rats were selected for the experiment, with an equal number of males and females, weighing approximately 180-230g.

[0097] Three rats were taken, and the backs of each rat were marked with four sites. Each group was injected with the solution described in the example.

[0098] 1. Fillers of Example 2, Comparative Example 1 and Comparative Example 2.

[0099] The injection method is as follows: Take 0.3 ml of the well-mixed filler solution from Experiment 1 and implant it subcutaneously into the back of the rat. After 8 weeks, take samples for MASSON staining to observe collagen formation.

[0100] The collagen regeneration stimulating ability of the fillers in each embodiment and comparative example is as follows: Figure 4 As shown,

[0101] Under Masson staining in Examples 1 and 2, abundant collagen fibers were observed around and in the related areas of the polylactic acid (PLA) microspheres within the tissue. These collagen fibers were widely distributed, occupying a high proportion of the field of view, and stained a deep blue, indicating abundant collagen fiber content and good maturity. The fibers were densely arranged, with some areas exhibiting interwoven or bundle-like structures. The integration and distribution with the surrounding tissue showed significant proliferation and deposition of collagen fibers after 8 weeks of PLA microsphere filling, indicating significant overall collagen regeneration. This may reflect a certain promoting effect of PLA microspheres on collagen formation. Under Masson staining in Comparative Example 1, numerous abnormal vacuolar structures were observed with a disordered distribution. The surrounding collagen fibers were sparser and more disordered than in Examples 1, with lighter staining and insufficient regularity and continuity. Under Masson staining in Comparative Example 2, many vacuolar structures were observed within the tissue, with relatively sparse distribution of surrounding collagen fibers, shallower staining depth, and poor regularity and continuity. Some areas showed broken and loose collagen fibers, indicating limited overall collagen proliferation and maturation. By comparing Examples 1 and 2 with Comparative Examples 1 and 2, it can be seen that the composite porous microspheres can promote collagen regeneration in advance and achieve a filling effect more quickly.

Claims

1. An injectable filler that can persistently suspend and stimulate collagen regeneration, characterized in that, The filler is mainly composed of porous composite microspheres and hydrogel; the porous composite microsphere material is a composite microsphere of porous polylactic acid microspheres and hydroxyapatite nanospheres; the hydrogel includes matrix material, water, isotonic agent, pH adjuster, and analgesic.

2. The injectable filler for persistent suspension and stimulation of collagen regeneration according to claim 1, characterized in that, The porous composite microspheres have a polylactic acid to hydroxyapatite mass ratio of 3:1 to 5:1 and a porous composite microsphere to matrix mass ratio of 1:30 to 1:

10.

3. The injectable filler for persistent suspension and stimulation of collagen regeneration according to claim 1, characterized in that, The porous composite microspheres are spherical or near-spherical, with a particle size of 10–120 μm and a pore size of 0.001–10 μm; preferably, the particle size of the porous composite microspheres is 40–80 μm and the pore size is 2–10 μm.

4. The injectable filler for persistent suspension and stimulation of collagen regeneration according to claim 1, characterized in that, The injectable filler that can persistently suspend and stimulate collagen regeneration according to claim 1 is characterized in that the porous polylactic acid microspheres have a particle size of 40-80 μm, and the pore size is preferably 2-10 μm.

5. The injectable filler for persistent suspension and stimulation of collagen regeneration according to claim 1, characterized in that, The hydroxyapatite nanospheres have a particle size of 0.1–5 μm and a pore size of 1–100 nm.

6. The injectable filler for persistent suspension and stimulation of collagen regeneration according to claim 1, characterized in that, The hydrogel includes a matrix material, water, an isotonic agent, a pH adjuster, and an analgesic.

7. The matrix material according to claim 6 is one or both of silk fibroin and collagen.

8. The method for preparing the injectable filler that can persistently suspend and stimulate collagen regeneration according to claim 1, characterized in that, Includes the following steps: Methods for preparing porous polylactic acid microspheres include emulsification. Methods for preparing porous composite microspheres include mineralization. The hydrogel was prepared by chemical cross-linking. The porous composite microspheres are added to the hydrogel and stirred thoroughly to obtain the final product.

9. The method for preparing the injectable filler that can persistently suspend and stimulate collagen regeneration according to claim 8, characterized in that, Includes the following steps: (1) Preparation of porous polylactic acid microspheres: Polylactic acid (PLA) is dissolved in an organic solvent to form an oil phase; gelatin or ammonium bicarbonate is completely dissolved in water to form an inner aqueous phase; polyvinyl alcohol (PVA) is dissolved in water to form an outer aqueous phase. The inner aqueous phase is slowly added to the oil phase and emulsified at a rate of 3000–8000 rpm / min for 1–10 min to form a primary emulsion; the outer aqueous phase is slowly added to the primary emulsion and emulsified at a rate of 1500–4000 rpm / min for 3–40 min; the mixture is then vacuum stirred at 100–600 rpm / min for 6–24 h to solidify; after the organic solvent in the solution has evaporated, the mixture is washed, dried, and passed through a sieve to obtain porous PLA microspheres. (2) Preparation of porous composite microspheres: Porous polylactic acid microspheres were immersed in a solution containing 0.05–0.2 M calcium chloride and 0.01–0.05 M magnesium chloride for 1–10 minutes. After immersion, they were removed and rinsed for 1–10 minutes. Then, they were immersed in a solution containing 0.05–0.2 M disodium hydrogen phosphate and 0.01–0.05 M sodium hydrochloride for 1–10 minutes. After immersion, they were removed and rinsed for 1–10 minutes. This alternating immersion process was repeated 2–4 times to complete the pre-mineralization. Sodium chloride, dipotassium hydrogen phosphate, magnesium chloride, 4-hydroxyethylpiperazine ethanesulfonic acid, calcium chloride, and sodium bicarbonate were mixed in a weight ratio of 22:1:1:12:1:2 to prepare a mixed solution. The pH of the mixed solution was adjusted to 6.4-7.2, and the solution was stirred until clear to form a mineralized solution. The pre-mineralized porous polylactic acid microspheres were placed in a mineralization solution and kept at 37°C for 1–2 days. After the solution was removed, the microspheres were filtered, washed, and dried to obtain porous composite microspheres. (3) Preparation of hydrogel: Weigh one or two of collagen and silk fibroin, mix with water and isotonic agent, and the concentration is 0.5% to 2%. (4) Mixing: Add the porous composite microspheres to the hydrogel, stir and mix thoroughly, then immediately dispense, freeze dry and sterilize by irradiation to obtain the filler.

10. The application of the injectable filler that can persistently suspend and stimulate collagen regeneration according to claim 1 in medical aesthetics, plastic surgery, filling and tissue defect repair.

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