Application of polylactic acid and its copolymers in the preparation of products that promote hair follicle growth and / or repair hair follicle damage

Abstract

This invention discloses the use of polylactic acid (PLA) and its copolymers in the preparation of products that promote hair follicle growth and repair, belonging to the field of biomedical technology. Through in vitro and in vivo experiments, this invention demonstrates that PLA can significantly upregulate the expression of lactate dehydrogenase A and transforming growth factor β2 in dermal papilla cells, promoting dermal papilla cell proliferation and hair follicle growth and damage repair. Based on these findings, this invention provides the application of PLA and its copolymers in the preparation of products that promote hair follicle growth and damage repair, offering a safe and effective treatment strategy for hair follicle-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of polylactic acid and its copolymers in the preparation of products that promote hair follicle growth and / or repair hair follicle damage. Background Technology

[0002] A hair follicle is a sac-like tissue at the root of a hair. Its structure is divided into upper and lower parts by a protrusion. The upper part, including the infundibulum and isthmus, is called the permanent component and does not participate in circulation or regeneration. The lower part, including the protuberance, hair bulb, and dermal papilla, accounts for about one-third of the hair follicle and is called the circulating component, participating in the regeneration cycle of the hair follicle. From the inside out, the layered structure of the hair follicle includes the inner root sheath, outer root sheath, and fibrous sheath.

[0003] Among the many cellular components of the hair follicle, dermal papilla cells (DPCs) play a crucial regulatory role. Originating from mesodermal-derived dermal tissue, DPCs aggregate to form the dermal papilla structure, which connects to the hair bulb. As the earliest identified type of stem cell regulator of hair follicles, DPCs determine the size and duration of hair follicle growth. During the morphogenesis stage of the hair follicle, embryonic dermal mesenchymal stem cells aggregate under the hair follicle basal plate to form dermal coagulation, which later develops into the dermal papilla. Through the establishment of a signal activation gradient with the epidermis via reciprocal signals, they promote the formation of hair follicle stem cells.

[0004] In the hair follicle cycle, dermal papilla cells are key signaling centers: during the resting phase, they secrete inhibitory signals such as BMPs to maintain the quiescent state of hair follicle stem cells; during the transition to the anagen phase, they accumulate activating signals such as FGFs, BMP inhibitors, and TGF-β2 to induce regeneration; during the transition from the anagen to the resting phase, they induce programmed cell death of basal epithelial cells through TGF-β signaling and Wnt antagonists. During aging, the dermal papilla undergoes a decrease in cell number and miniaturization, leading to a reduction in regulatory signals and a progressive loss of the dermal stem cells that maintain its number, all contributing to the aging of the hair follicle.

[0005] Hair follicles, as complex micro-organs, rely on precise intercellular communication and cyclical regeneration for normal function. However, our understanding of the pathological mechanisms of hair follicle-related diseases is currently insufficient, posing challenges to the clinical translation of treatment strategies targeting structural abnormalities or functional disorders of the hair follicles themselves. Therefore, developing effective and highly targeted treatments based on the pathological regulatory mechanisms of hair follicles has become an urgent problem to be solved in this field.

[0006] Existing treatments for hair follicle growth disorders suffer from significant side effects and poor targeting. While biodegradable biomaterials are widely used in the medical field, their mechanisms and applications as active ingredients to directly promote hair follicle growth have not been reported. This invention addresses this gap by providing a novel strategy for promoting hair follicle growth based on polylactic acid (PLA). Summary of the Invention

[0007] In recent years, biodegradable biomedical polymer materials have been widely used in surgical sutures, surgical adhesives, and drug release materials due to their high biocompatibility, good biocompatibility, and ability to be absorbed through human metabolism.

[0008] However, the aforementioned biodegradable biomedical polymers have not yet been found to be used as active ingredients to promote hair follicle growth, and their mechanisms of action in promoting dermal papilla cell (DPC) proliferation, upregulating lactate dehydrogenase A expression in DPCs, and promoting TGF-β2 secretion have not been elucidated.

[0009] To address the problems existing in the prior art, the present invention provides the application of polylactic acid and its copolymers in the preparation of products that promote hair follicle growth.

[0010] Specifically, the present invention includes the following: The polylactic acid and / or polylactic acid copolymer has the following effects: 1) Promotes the proliferation of dermal papilla cells (DPCs); 2) Upregulates lactate dehydrogenase A expression in DPCs; 3) Promotes the secretion of TGF-β2 in DPCs; 4) Promotes hair follicle growth.

[0011] Based on the above effects, polylactic acid and its copolymers can be used to prepare the following products: 1) Prepare products for treating hair follicle dysfunction characterized by limited function and / or reduced number of hair papilla cells; 2) Prepare products for treating degenerative hair follicle diseases characterized by hair follicle cycle disorders; 3) Prepare products to repair hair follicle damage caused by physical, chemical or inflammatory factors.

[0012] In some embodiments, the polylactic acid and / or polylactic acid copolymer is prepared as microparticles, preferably microspheres, and more preferably porous microspheres. The porosity of the porous microspheres is 10% to 95%, preferably 50% to 80%.

[0013] In some embodiments, the molecular weight of the polylactic acid and / or polylactic acid copolymer is 400 Da to 50 kDa, preferably, the molecular weight of the polylactic acid and / or polylactic acid copolymer is 2 kDa to 10 kDa.

[0014] In some embodiments, the size of the particles is 50 nm to 100 μm; preferably, the size of the particles is 10 μm to 50 μm; more preferably, the size of the particles is 15 μm to 35 μm.

[0015] In some embodiments, the polylactic acid includes one or more of L-polylactic acid and racemic polylactic acid; preferably, the polylactic acid is L-polylactic acid.

[0016] In some embodiments, the polylactic acid copolymer includes one or more of polylactic acid-glycolic acid copolymer, polylactic acid-polyethylene glycol copolymer, polyethylene glycol-polylactic acid-glycolic acid copolymer, and lactide-caprolactone copolymer.

[0017] In some embodiments, the L-lactic acid content in the polylactic acid and / or polylactic acid copolymer is at least 20%, for example at least about 25%, for example at least about 30%, for example at least about 35%, for example at least about 40%, for example at least about 45%, for example at least about 50%, for example at least about 55%, for example at least about 60%, for example at least about 65%, for example at least about 70%, for example at least about 75%, for example at least about 80%, for example at least about 85%, for example at least about 90%, for example at least about 95%. It should be understood that the percentages mentioned correspond to mass percentages (w / w).

[0018] According to an embodiment of the present invention, the polylactic acid and / or polylactic acid copolymer microparticles can be obtained by methods such as nanoprecipitation, emulsification dissolution and volatilization, spray drying, and physical crushing.

[0019] In some embodiments, the polylactic acid and / or polylactic acid copolymer porous microspheres can be prepared by an emulsification-solvent evaporation method, the preparation steps of which include: Step 1: Prepare the solution: (1) A polymer solution is prepared by dissolving polylactic acid and / or polylactic acid copolymers in an organic solvent, wherein the polymer concentration is 0.5% to 5% (w / v). Preferred organic solvents are one or more of dichloromethane, chloroform, ethyl acetate, and acetone. Preferably, the polymer concentration is 1% to 3%.

[0020] (2) Prepare an aqueous solution of polyvinyl alcohol as the aqueous phase with a concentration of 0.1% to 2% (w / v), and optionally add a porogen with a concentration of 5% to 20% (w / v), such as sodium chloride. Preferably, the concentration of polyvinyl alcohol is 0.5% to 1%.

[0021] Step 2: Form the emulsion and allow it to solidify. At a shear rate of 100 rpm to 10,000 rpm, the polymer solution from step (1) is added to the aqueous phase from step (2) and emulsified for 5 to 20 minutes to form an emulsion. Subsequently, the mixture is stirred continuously at a stirring rate of 200 rpm to 500 rpm for 2 to 4 hours to allow the organic solvent to evaporate completely and the polymer to solidify into microparticles.

[0022] Step 3, Post-processing: The obtained microparticles are collected by centrifugation or filtration, washed with deionized water to remove surfactants and water-soluble porogens, and finally freeze-dried to obtain the porous microspheres.

[0023] By adjusting the parameters in the above steps, this invention can obtain porous microspheres with different particle sizes: the stirring speed is the key parameter for controlling the particle size. Specifically, a stirring speed of 100 rpm to 800 rpm is suitable for preparing microspheres with a particle size of 20 μm to 100 μm; 800 rpm to 3,000 rpm is suitable for preparing microspheres with a particle size of 1 μm to 20 μm; and 3,000 rpm to 10,000 rpm is suitable for preparing microspheres with a particle size of 50 nm to 1 μm.

[0024] Polymer concentration is a key parameter for controlling pore structure. Low concentrations of 0.5% to 2% are conducive to the formation of porous structures with high porosity, which can reach 50% to 85%.

[0025] By selectively adding and adjusting the concentration of porogens (such as sodium chloride), the porosity and pore size of the microspheres can be further adjusted to achieve a porosity of 10% to 95%, with a preferred porosity range of 50% to 80%.

[0026] In some embodiments, the polylactic acid and / or polylactic acid copolymers, when administered, contain 0.1% to 80% polylactic acid and its copolymers, polylactic acid microparticles, and polylactic acid copolymer microparticles, more preferably 0.5%, 1%, and 2%.

[0027] In some embodiments, the drug may optionally further include excipients, said excipients including at least one of stabilizers, fillers, binders and lubricants; Preferably, the stabilizer is selected from one or two of carboxymethyl cellulose and mannitol; Preferably, the filler is selected from one or more of lactose, mannitol, cyclodextrin, and sorbitol; Preferably, the adhesive is selected from one or more of hydroxypropyl cellulose, methylcellulose, sodium hyaluronate, collagen, and polyvinylpyrrolidone; Preferably, the lubricant is selected from one or more of magnesium stearate, calcium stearate, and stearic acid.

[0028] In some embodiments, the drug is administered via local administration, such as by injection, microneedle delivery, or ointment / gel application, directly to the target area of ​​the skin. It can also be formulated as a nanoparticle suspension for easy transdermal delivery.

[0029] This invention is the first to demonstrate through in vitro cell experiments that polylactic acid (PLA) and its copolymers can upregulate the expression of LDHA and TGF-β2 in dermal papilla cells of hair follicles. Based on this, tissue section analysis further verified the effects of PLA and its copolymers on promoting hair follicle growth and repair. The beneficial effects of this invention are: This invention uses biodegradable PLA materials (especially porous microspheres with a particle size of 10-50 μm) directly as active ingredients, acting on hair follicles through injection and other methods, which can target and promote the proliferation of dermal papilla cells and the expression of related repair factors, thereby effectively promoting hair follicle growth. This invention provides a novel, safe, and effective material treatment strategy with a clear mechanism for hair follicle damage, degenerative hair follicle diseases, and other hair follicle-related diseases. Attached Figure Description

[0030] Figure 1 These are images from immunofluorescence analysis of cells using ALP and Versican antibodies. Figure 1 A represents dermal papilla cells. Figure 1 B represents fibroblasts.

[0031] Figure 2 These are the immunofluorescence analysis results of DPC cells labeled with Ki67 antibody in groups A through E. Figure 2 Image A is a representative image from an immunofluorescence analysis of cells. Figure 2 B is the correct answer. Figure 2 Statistical analysis of data A. mean ± SEM; *p<0.05; **p<0.01; ****p<0.0001.

[0032] Figure 3 These are the immunofluorescence analysis results of DPC cells labeled with Ki67 antibody in groups A, F, and D. Figure 3 Image A is a representative image from an immunofluorescence analysis of cells. Figure 3 B is the correct answer. Figure 3 Statistical analysis of data A. mean ± SEM; *p<0.05; ****p<0.0001.

[0033] Figure 4 These are the immunofluorescence analysis results of EdU-labeled DPC cells in groups A through E. Figure 4 Image A is a representative image from an immunofluorescence analysis of cells. Figure 4 B is the correct answer. Figure 4 Statistical analysis of data A. mean ± SEM; **p<0.01; ***p<0.001; ****p<0.0001.

[0034] Figure 5 These are the immunofluorescence analysis results of EdU-labeled DPC cells in groups A, F, and D. Figure 5Image A is a representative image from an immunofluorescence analysis of cells. Figure 5 B is the correct answer. Figure 5 Statistical analysis of data A. mean ± SEM; **p<0.01; ****p<0.0001.

[0035] Figure 6 This is a graph showing the results of Ldha protein expression level detection in DPC cells. Figure 6 Figure A shows the results of Western blot analysis of Ldha protein levels in DPC cells from groups A to E. Figure 6 B is the correct answer. Figure 6 Statistical analysis of the relative expression level of Ldha protein in A; Figure 6 Figure C shows the results of Western blot analysis of Ldha protein levels in DPC cells from groups A, F, and D. Figure 6 D is the correct answer. Figure 6 Statistical analysis of the relative expression level of Ldha protein in C.

[0036] Figure 7 This is a graph showing the Ldha mRNA levels in DPC cells. Figure 7 A represents the Ldha mRNA level analysis of DPC cells in groups A through E. Figure 7 B represents the Ldha mRNA level analysis of DPC cells in groups A, F, and D. mean ± SEM; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0037] Figure 8 This is a graph showing the results of TGF-β2 expression level detection in DPC cells. Figure 8 A represents the analysis of TGF-β2 protein secretion levels detected by ELISA. Figure 8 B represents the TGF-β2 mRNA level analysis. mean ± SEM; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0038] Figure 9 This is a picture of the hair follicle status test results. Figure 9 A is a representative image of skin tissue sections stained with hematoxylin and eosin (HE). Figure 9 B represents a statistical analysis of the number of hair follicles in groups A through E. Figure 9 C represents the statistical analysis of the average hair follicle diameter in groups A through E. mean ± SEM; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Detailed Implementation

[0039] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0040] Preparation Example 1: Low molecular weight polylactic acid (PLLA) small particle size high porosity porous microspheres Polymer: Polylactic acid (PLLA), molecular weight 2 kDa.

[0041] Preparation method: Solution preparation: Dissolve 0.1 g PLLA in 5 mL of dichloromethane to prepare a 2% (w / v) oil phase solution. The aqueous phase is 50 mL of an aqueous solution containing 1% (w / v) polyvinyl alcohol and 20% (w / v) sodium chloride.

[0042] Emulsification: The oil phase solution is rapidly injected into the aqueous phase at a high speed of 10,000 rpm in a high-speed homogenizer, and the homogenization is carried out at high speed for 2 minutes to form a primary emulsion.

[0043] Curing into spheres: Transfer the emulsion to a magnetic stirrer and stir at 300 rpm at room temperature for 4 hours to allow the organic solvent dichloromethane to completely evaporate and the polymer to cure.

[0044] Post-processing: The microspheres were collected by centrifugation at 8000 rpm for 10 minutes, washed three times with deionized water to remove residual surfactants and salts, and then freeze-dried to obtain dried PLLA porous microspheres.

[0045] The obtained porous microspheres have an average particle size of 8 μm and a porosity of approximately 80%.

[0046] Preparation Example 2: High molecular weight polylactic acid (PLLA) large particle size low porosity porous microspheres Polymer: Polylactic acid (PLLA), molecular weight 50 kDa.

[0047] Preparation method: Solution preparation: Dissolve 0.5 g PLLA in 10 mL of dichloromethane to prepare a 5% (w / v) oil phase solution. The aqueous phase is 100 mL of an aqueous solution containing 1% (w / v) polyvinyl alcohol and 5% (w / v) sodium chloride.

[0048] Emulsification: The oil phase solution was slowly added dropwise to the aqueous phase at 300 rpm using a magnetic stirrer. After the addition was complete, the mixture was stirred at 300 rpm for 15 minutes to emulsify.

[0049] Solidify into spheres: Maintain a stirring speed of 300 rpm and stir at room temperature for 3 hours to allow the solvent to evaporate completely.

[0050] Post-processing: Collect microspheres by centrifugation at 5000 rpm for 5 minutes, wash three times with deionized water, and freeze-dry.

[0051] The obtained porous microspheres have an average particle size of 90 μm and a porosity of approximately 15%.

[0052] Preparation Example 3: Medium molecular weight polylactic acid (PLLA) medium particle size high porosity porous microspheres

[0053] Polymer: Polylactic acid (PLLA), molecular weight 10 kDa.

[0054] Preparation method: Solution preparation: Dissolve 0.2 g PLLA in 10 mL of dichloromethane to prepare a 2% (w / v) oil phase solution. The aqueous phase is 150 mL of an aqueous solution containing 0.5% (w / v) polyvinyl alcohol and 15% (w / v) sodium chloride.

[0055] Emulsification: The oil phase solution was added dropwise to the aqueous phase at a mechanical stirrer speed of 1200 rpm and emulsified for 10 minutes.

[0056] Solidify into spheres: Reduce the stirring speed to 400 rpm and stir at room temperature for 3.5 hours.

[0057] Post-processing: Collect microspheres by centrifugation at 6000 rpm for 8 minutes, wash three times with deionized water, and freeze-dry.

[0058] The obtained porous microspheres have an average particle size of 25 μm and a porosity of approximately 70%.

[0059] Preparation Example 4: Low molecular weight polylactic acid (PLLA) medium-sized, medium-porosity porous microspheres Polymer: Polylactic acid (PLLA), molecular weight 2 kDa.

[0060] Preparation method: Solution preparation: Dissolve 0.1 g PLLA in 10 mL of dichloromethane to prepare a 1% (w / v) oil phase solution. The aqueous phase is 100 mL of an aqueous solution containing 1% (w / v) polyvinyl alcohol and 10% (w / v) sodium chloride.

[0061] Emulsification: The oil phase is added dropwise to the water phase at 800 rpm with a mechanical stirrer, and emulsification is carried out for 8 minutes.

[0062] Solidify into spheres: Adjust the stirring speed to 500 rpm and stir at room temperature for 4 hours.

[0063] Post-processing: Collect microspheres by centrifugation at 7000 rpm for 7 minutes, wash and freeze-dry.

[0064] Product characteristics: The obtained porous microspheres have an average particle size of 15 μm and a porosity of approximately 50%.

[0065] Preparation Example 5: High molecular weight polylactic acid (PLLA) medium-sized, low-porosity porous microspheres Polymer: Polylactic acid (PLLA), molecular weight 50 kDa.

[0066] Preparation method: Solution preparation: Dissolve 0.3 g PLLA in 10 mL of dichloromethane to prepare a 3% (w / v) oil phase solution. The aqueous phase is 100 mL of an aqueous solution containing 1% (w / v) polyvinyl alcohol and 8% (w / v) sodium chloride.

[0067] Emulsification: The oil phase was added dropwise to the water phase at 1000 rpm with a mechanical stirrer and emulsified for 12 minutes.

[0068] Solidify into spheres: Maintain a stirring speed of 1000 rpm and stir at room temperature for 3 hours.

[0069] Post-processing: Collect microspheres by centrifugation at 6000 rpm for 8 minutes, wash and freeze-dry.

[0070] The obtained porous microspheres have an average particle size of 35 μm and a porosity of approximately 25%.

[0071] Preparation Example 6: Small-particle-size, high-porosity porous microspheres of low molecular weight polylactic-co-glycolic acid copolymer (PLGA) Polymer: Polylactic acid-glycolic acid copolymer (PLGA, LA:GA=50:50), molecular weight 2 kDa, intrinsic viscosity 0.15 dL / g.

[0072] Preparation method: Solution preparation: Dissolve 0.15 g PLGA in 10 mL of ethyl acetate to prepare a 1.5% (w / v) low-concentration oil phase solution. The aqueous phase is 100 mL of an aqueous solution containing 1% (w / v) polyvinyl alcohol and 22% (w / v) sodium chloride.

[0073] Emulsification: The oil phase is rapidly injected into the aqueous phase at a high shear speed of 12,000 rpm in a high-speed homogenizer, and homogenization is carried out at high speed for 1.5 minutes to form fine emulsion droplets.

[0074] Curing into spheres: Transfer the emulsion to a magnetic stirrer and stir at 250 rpm at room temperature for 5 hours (ethyl acetate has a high boiling point and evaporates slowly) to cure the polymer.

[0075] Post-processing: Collect microspheres by centrifugation at 8500 rpm for 10 minutes, wash and freeze-dry.

[0076] The obtained PLGA porous microspheres have an average particle size of approximately 8 μm and a porosity of approximately 75%.

[0077] Preparation Example 7: Medium molecular weight polylactic-co-glycolic acid copolymer (PLGA) medium particle size high porosity porous microspheres Polymer: Polylactic acid-glycolic acid copolymer (PLGA, LA:GA=50:50), molecular weight 10 kDa. Intrinsic viscosity 0.2 dL / g.

[0078] Preparation method: Solution preparation: Dissolve 0.25 g PLGA in 10 mL of ethyl acetate to prepare a 2.5% (w / v) oil phase solution. The aqueous phase is 100 mL of an aqueous solution containing 0.8% (w / v) polyvinyl alcohol and 18% (w / v) sodium chloride.

[0079] Emulsification: The oil phase is added dropwise to the water phase at a mechanical stirrer speed of 1500 rpm, and emulsification is carried out for 6 minutes.

[0080] Solidification into spheres: Reduce the stirring speed to 350 rpm and stir at room temperature for 5 hours (ethyl acetate has a high boiling point and evaporates slowly).

[0081] Post-processing: Collect microspheres by centrifugation at 6500 rpm for 9 minutes, wash and freeze-dry.

[0082] The obtained porous microspheres have an average particle size of 18 μm and a porosity of approximately 65%.

[0083] Preparation Example 8: High molecular weight polylactic-co-glycolic acid copolymer (PLGA) large-particle-size, low-porosity porous microspheres Polymer: Polylactic acid-glycolic acid copolymer (PLGA, LA:GA=50:50), molecular weight 50 kDa. Intrinsic viscosity 0.4 dL / g.

[0084] Preparation method: Solution preparation: Dissolve 0.6 g PLGA in 10 mL of ethyl acetate to prepare a 6% (w / v) oil phase solution. The aqueous phase is 100 mL of an aqueous solution containing 0.8% (w / v) polyvinyl alcohol and 3% (w / v) sodium chloride.

[0085] Emulsification: The oil phase was added dropwise to the water phase at a low shear speed of 250 rpm using a magnetic stirrer, and emulsified for 20 minutes.

[0086] Solidification into spheres: Maintain a stirring speed of 250 rpm and stir at room temperature for 6 hours (ethyl acetate has a high boiling point and evaporates slowly).

[0087] Post-processing: Collect microspheres by centrifugation at 4500 rpm for 5 minutes, wash and freeze-dry.

[0088] The obtained porous microspheres have an average particle size of 70 μm and a porosity of approximately 15%.

[0089] Preparation Example 9: Racemic Polylactic Acid (PDLLA) Porous Microspheres Polymer: racemic polylactic acid (PDLLA), molecular weight 15 kDa.

[0090] Preparation method: Solution preparation: Dissolve 0.2 g PDLLA in 10 mL of chloroform to prepare a 2% (w / v) oil phase solution. The aqueous phase is 150 mL of an aqueous solution containing 1% (w / v) polyvinyl alcohol and 12% (w / v) sodium chloride.

[0091] Emulsification: The oil phase was injected into the aqueous phase at a high speed of 5000 rpm in a high-speed homogenizer and homogenized for 1 minute.

[0092] Solidification into spheres: Transfer the emulsion to a magnetic stirrer and stir at 400 rpm at room temperature for 4 hours.

[0093] Post-processing: Collect microspheres by centrifugation at 8000 rpm for 10 minutes, wash three times with deionized water, and freeze-dry.

[0094] The obtained porous microspheres have an average particle size of 20 μm and a porosity of approximately 60%.

[0095] Preparation Example 10: Porous Microspheres of Polylactic Acid-Polyethylene Glycol Copolymer (mPEG-PLLA) Polymer: Polylactic acid-polyethylene glycol amphiphilic block copolymer (mPEG-PLLA, PEG segment molecular weight 2kDa, PLLA segment molecular weight 8kDa).

[0096] Preparation method: Solution preparation: Dissolve 0.15 g mPEG-PLLA in 8 mL of dichloromethane to prepare an oil phase solution of approximately 1.9% (w / v). The aqueous phase is 80 mL of deionized water, in which 10% (w / v) sodium chloride is dissolved (no PVA is added, utilizing the emulsifying properties of the copolymer itself).

[0097] Emulsification: The oil phase is slowly dripped into the water phase at 600 rpm using a magnetic stirrer. After the addition is complete, continue stirring and emulsifying for 20 minutes.

[0098] Solidify into spheres: Maintain a stirring speed of 600 rpm and stir at room temperature for 4 hours.

[0099] Post-processing: Collect microspheres by centrifugation at 7000 rpm for 7 minutes, wash three times with deionized water, and freeze-dry.

[0100] The obtained porous microspheres have an average particle size of 30 μm and a porosity of approximately 55%.

[0101] Preparation Example 11: Polyethylene glycol-polylactic acid-glycolic acid copolymer (PEG-PLGA) porous microspheres Polymer: Polyethylene glycol-polylactic acid-glycolic acid triblock copolymer (PEG-PLGA, PEG segment molecular weight 1.5kDa, PLGA segment LA:GA=75:25).

[0102] Preparation method: Solution preparation: Dissolve 0.18 g PEG-PLGA in 9 mL of a mixed solvent of dichloromethane and acetone (volume ratio 4:1) to prepare a 2% (w / v) oil phase solution. The aqueous phase is 120 mL of an aqueous solution containing 0.3% (w / v) polyvinyl alcohol and 14% (w / v) sodium chloride.

[0103] Emulsification: The oil phase is added dropwise to the water phase at a mechanical stirrer speed of 900 rpm, and emulsification is carried out for 10 minutes.

[0104] Solidify into spheres: Adjust the stirring speed to 450 rpm and stir at room temperature for 4 hours.

[0105] Post-processing: Collect microspheres by centrifugation at 7500 rpm for 6 minutes, wash and freeze-dry.

[0106] The obtained porous microspheres have an average particle size of 22 μm and a porosity of approximately 58%.

[0107] Preparation Example 12: Porous microspheres of lactide-caprolactone copolymer (P(LA-co-CL)) Polymer: Lactide-caprolactone random copolymer (P(LA-co-CL), LA:CL=70:30), molecular weight 25 kDa.

[0108] Preparation method: Solution preparation: Dissolve 0.22 g P(LA-co-CL) in 11 mL of dichloromethane to prepare a 2% (w / v) oil phase solution. The aqueous phase is 110 mL of an aqueous solution containing 1% (w / v) polyvinyl alcohol and 16% (w / v) sodium chloride.

[0109] Emulsification: The oil phase is rapidly injected into the aqueous phase at a high speed of 3000 rpm in a high-speed homogenizer, and homogenized for 30 seconds.

[0110] Solidification into spheres: Transfer the emulsion to a magnetic stirrer and stir at 500 rpm at room temperature for 3.5 hours.

[0111] Post-processing: Collect microspheres by centrifugation at 6000 rpm for 8 minutes, wash and freeze-dry.

[0112] The obtained porous microspheres have an average particle size of 12 μm and a porosity of approximately 72%.

[0113] Preparation Example 13: Small-diameter solid microspheres of low molecular weight polylactic acid (PLLA) Polymer: Polylactic acid (PLLA), molecular weight 2 kDa.

[0114] Preparation method: Solution preparation: Dissolve 0.1 g PLLA in 5 mL of dichloromethane to prepare a 2% (w / v) oil phase solution. The aqueous phase is 50 mL of an aqueous solution containing 1% (w / v) polyvinyl alcohol (without adding sodium chloride porogen).

[0115] Emulsification: The oil phase solution is rapidly injected into the aqueous phase at a high speed of 10,000 rpm in a high-speed homogenizer, and the homogenization is carried out at high speed for 2 minutes to form a primary emulsion.

[0116] Curing into spheres: Transfer the emulsion to a magnetic stirrer and stir at 300 rpm at room temperature for 4 hours to allow the organic solvent dichloromethane to completely evaporate and the polymer to cure.

[0117] Post-processing: The microspheres were collected by centrifugation at 8000 rpm for 10 minutes, washed three times with deionized water to remove residual surfactants, and then freeze-dried to obtain dry PLLA solid microspheres.

[0118] The resulting solid microspheres had an average particle size of about 8 μm and extremely low porosity (close to 0), which was consistent with the porous microspheres prepared in Example 1 in terms of particle size and molecular weight.

[0119] Preparation Example 14: Small-size solid microspheres of low molecular weight polylactic-co-glycolic acid copolymer (PLGA) Polymer: Polylactic acid-glycolic acid copolymer (PLGA, LA:GA=50:50), molecular weight 2 kDa, intrinsic viscosity 0.15 dL / g.

[0120] Preparation method: Solution preparation: Dissolve 0.15 g PLGA in 10 mL ethyl acetate to prepare a 1.5% (w / v) low-concentration oil phase solution. The aqueous phase is 100 mL of an aqueous solution containing 1% (w / v) polyvinyl alcohol (without adding sodium chloride porogen).

[0121] Emulsification: The oil phase solution is rapidly injected into the aqueous phase at a high speed of 10,000 rpm in a high-speed homogenizer, and the homogenization is carried out at high speed for 2 minutes to form a primary emulsion.

[0122] Curing into spheres: Transfer the emulsion to a magnetic stirrer and stir at 300 rpm at room temperature for 4 hours to allow the organic solvent to evaporate completely and the polymer to cure.

[0123] Post-processing: The microspheres were collected by centrifugation at 8000 rpm for 10 minutes, washed three times with deionized water to remove residual surfactant, and then freeze-dried to obtain dried PLGA solid microspheres. The obtained solid microspheres had an average particle size of approximately 8 μm and extremely low porosity (close to 0), consistent with the porous microspheres of Preparation Example 6 in terms of particle size and molecular weight.

[0124] Preparation Example 15: Preparation of polylactic acid or its copolymer microparticles by spray drying Polymers: Polylactic acid (PLLA), polylactic acid-glycolic acid copolymer (PLGA), polylactic acid-polyethylene glycol copolymer (mPEG-PLLA), lactide-caprolactone random copolymer (P(LA-co-CL), polyethylene glycol-polylactic acid-glycolic acid triblock copolymer (PEG-PLGA), molecular weight 2kDa.

[0125] Preparation method: Weigh 2.0 g of any one of the above polymer raw materials, dissolve it in 100 mL of dichloromethane, and stir magnetically for 30 minutes until completely dissolved to prepare a 2% (w / v) clear solution. Prepare the solution using a small spray dryer with the following process parameters: inlet temperature 60℃, feed rate 3 mL / min, spray pressure 0.15 MPa, and exhaust fan power 100%. The resulting microparticles are collected by a cyclone separator and vacuum dried at 40℃ for 24 hours to remove residual solvent. The microparticles obtained under these conditions are spherical with a particle size range of 10–50 μm; by adjusting the parameters (concentration 2.5%, spray pressure 0.12 MPa), narrowly distributed microparticles of 15–35 μm can be obtained.

[0126] Preparation Example 16: Preparation of polylactic acid or its copolymer microparticles by physical crushing method Polymers: Polylactic acid (PLLA), polylactic acid-glycolic acid copolymer (PLGA), polylactic acid-polyethylene glycol copolymer (mPEG-PLLA), lactide-caprolactone random copolymer (P(LA-co-CL), polyethylene glycol-polylactic acid-glycolic acid triblock copolymer (PEG-PLGA), molecular weight 2kDa.

[0127] Weigh 50g of any one of the above polymer raw materials, spread it evenly in a stainless steel pan, and immerse it in liquid nitrogen for pre-freezing for 5 minutes to fully embrittle it. Quickly transfer it to a cryogenic grinder and grind it four times at 20,000 rpm at -196℃ for 30 seconds each time, with a 1-minute interval to prevent temperature rise. The resulting coarse powder is classified using a vibrating sieve, passing it through 200-mesh and 400-mesh sieves in sequence to collect irregularly shaped particles with a particle size of 15~35μm.

[0128] Example 1: Identification of hair papilla cell strains 1. Experimental methods and procedures: 1) Cell resuscitation: Mouse dermal papilla cells (MDPC) were purchased from Qingqi (Shanghai) Biotechnology Development Co., Ltd. as cryopreserved cells and transported on dry ice. The cryovials were directly immersed in 37°C warm water, agitated occasionally to thaw them quickly. The cryovials were removed, and the cell suspension was aspirated with a pipette, added to centrifuge tubes, and at least 5 times the volume of complete culture medium (pyruvate-free DMEM, 10% FBF, antibiotics) was added and mixed well. The cells were centrifuged at 1000 r / min for 10 min. The supernatant was discarded, the cell pellet at the bottom of the centrifuge tube was resuspended, the cells were counted, the cell density was adjusted, and the cells were seeded into 6 cm cell culture dishes and incubated statically at 37°C in a 5% CO2 incubator. The culture medium was changed the following day, and the cells were cultured continuously.

[0129] 2) Cell passage: Cells with 80% cell healing can be passaged. Aspirate the complete culture medium and gently wash three times with PBS buffer. Add 2 ml of 0.25% EDTA trypsin and digest for 2 min. Then wash the cells off with complete culture medium. Centrifuge at 1000 r / min for 10 min, discard the supernatant and collect the cells. Plate the cells into three 6 cm cell culture dishes and add 5 ml of complete culture medium to each.

[0130] 3) Immunofluorescence assay: Cells were seeded on round coverslips. When the cells reached 80% confluence, the culture medium was discarded, and PBS was added and incubated for 5 min. 4% PFA was added and incubated at room temperature for 15 min. The PFA was discarded, and PBS was added and incubated for 5 min. PBS containing 0.2% Triton X-100 was added and incubated for 10 min. The cells were washed three times with PBS, 5 min each time. PBS containing 2% BSA + 0.2% Triton X-100 was added and the cells were blocked for 1 h. Primary antibodies (Anti-ALP purchased from Santa Cruz, Anti-Versican purchased from Invitrogen) were added and incubated overnight at 4°C. The primary antibodies were discarded, and the cells were washed three times with PBS. The secondary fluorescent antibody (fluorescently conjugated secondary antibody purchased from Invitrogen) was added and incubated at room temperature for 1-2 h. The secondary antibody was discarded, and the cells were washed three times with PBS. DAPI was used to label the cell nuclei. After mounting and drying, the cells were photographed using a fluorescence confocal microscope.

[0131] 2. Experimental Results: Immunofluorescence detection results as follows Figure 1 As shown, DPC cells are positive for ALP and Versican (…). Figure 1 A), fibroblasts treated in the same way were selected as a negative control. Figure 1 B) indicates that the obtained DPC cell line is correct.

[0132] Example 2: Polylactic acid and its copolymers promote the proliferation of dermal papilla cells 1. Grouping and administration: Group A (control group): DMEM basal culture medium (purchased from HYCLONE); Group B (PLGA solid microspheres): DMEM basal medium containing 0.5 mg / mL PLGA solid microspheres (prepared in Preparation Example 14); Group C (mPEG-PLLA porous microspheres): DMEM basal medium containing 0.5 mg / mL mPEG-PLLA porous microspheres (prepared in Preparation Example 10).

[0133] Group D (low molecular weight PLGA porous microspheres): DMEM basal medium containing 0.5 mg / mL low molecular weight PLLA small particle size high porosity porous microspheres (prepared in Preparation Example 6).

[0134] Group E (PLLA porous microspheres): DMEM basal medium containing 0.5 mg / mL low molecular weight PLLA small particle size solid microspheres (prepared in Preparation Example 1).

[0135] Group F (high molecular weight PLGA porous microspheres): DMEM basal medium containing 0.5 mg / mL PLLA porous microspheres (prepared in Preparation Example 8).

[0136] 2. Cell immunofluorescence detection method and procedure (Ki67 labeling): 1) Seed cells on round coverslips. When the cells reached 80% confluence, DMEM basal medium and DMEM basal medium containing the corresponding 0.5 mg / mL microspheres were added to each group of cells respectively for 16 h of pretreatment. 2) Discard the culture medium and add PBS buffer for incubation for 5 min; 3) Add 4% PFA and incubate at room temperature for 15 min; 4) Discard the PFA and incubate with PBS buffer for 5 min; 5) Add PBS buffer containing 0.2% Triton X-100 and incubate for 10 min; 6) Wash three times with PBS buffer, 5 min each time; 7) Block with PBS buffer containing 2% BSA + 0.2% Triton X-100 for 1 h; 8) Add primary antibody (Anti-Ki67, purchased from abcam) and incubate overnight at 4°C (antibody dilution buffer and blocking buffer are the same). 9) Discard the primary antibody and wash three times with PBS buffer; 10) Add fluorescent secondary antibody (purchased from Invitrogen) and incubate at room temperature for 1-2 h (antibody dilution solution is PBS); 11) Discard the secondary antibody and wash three times with PBS buffer; 11) DAPI (purchased from Roche) was used to label cell nuclei; 12) After the slide is mounted and dried, it is photographed using a fluorescence confocal microscope.

[0137] 3. Cell immunofluorescence detection method and procedure (EdU labeling): 1) Seed cells on round coverslips. When the cells reached 80% confluence, DMEM basal medium and DMEM basal medium containing the corresponding 0.5 mg / mL microspheres were added to each group of cells respectively for 16 h of pretreatment. 2) Add EdU (Click-iT) to the culture medium TM EdU Imaging Kit (purchased from Thermo Fisher Scientific) was incubated at a final concentration of 10 μM for 45 min. 3) Remove the culture medium and add 4% PFA. Incubate at room temperature for 15 min. 4) Discard the PFA and wash twice with PBS containing 3% BSA; 5) Add PBS buffer containing 0.5% Triton X-100 and incubate at room temperature for 20 min; 6) Prepare Click-iT reaction buffer (100 μL Click-iT reaction buffer, 800 μL CuSO4, 100 μL 1× Click-iT reaction buffer additive) according to the kit instructions. 7) Remove the permeabilization solution and wash twice with PBS containing 3% BSA; 8) Add Click-iT reaction buffer and incubate in the dark for 30 minutes; 9) Remove the reaction buffer and wash twice with PBS containing 3% BSA; 10) DAPI labels the cell nucleus; 11) After mounting and drying, the slides are photographed using a fluorescence confocal microscope.

[0138] 4. Experimental Results: Ki67 is a nuclear protein that interacts with the outer regions of condensed chromosomes during cell division. This protein is expressed in the G1, S, G2, and M phases, but not in the G0 phase. Therefore, Ki67-positive cells represent cells within the cell cycle, that is, cells undergoing DNA, RNA, and protein synthesis or dividing, i.e., cells in a proliferating state.

[0139] The results are as follows Figure 2 , Figure 3The results showed that microsphere treatment in groups B through F increased the proportion of Ki67 positive cells. Among them, the proportion of Ki67 positive cells in group D (low molecular weight PLGA porous microspheres) increased significantly and was significantly better than that in group B (PLGA solid microspheres) and group F (high molecular weight PLGA porous microspheres).

[0140] To further confirm the promoting effect of polylactic acid and its copolymers on the proliferation of DPC cells, we used EdU to label pretreated DPC cells. EdU is an analogue of thymine and can be taken up into the nucleus of cells in the process of proliferation (S phase) and incorporated into the synthesized DNA strand, replacing the natural thymine deoxyribonucleotide, thereby labeling cells in the proliferating state.

[0141] The results are as follows Figure 4 , Figure 5 The results showed that EdU labeling was consistent with Ki67 labeling. After 16 h of treatment with microspheres in groups B to F, the proportion of EdU-positive cells increased significantly. The increase in the proportion of EdU-positive cells in group D (low molecular weight PLGA porous microspheres) was particularly significant and was significantly better than that in group B (PLGA solid microspheres) and group F (high molecular weight PLGA porous microspheres).

[0142] The above results indicate that PLLA, PLGA, and mPEG-PLLA all promoted the proliferation of dermal papilla cells, with PLGA porous microspheres exhibiting the best proliferative effect.

[0143] Example 3: Polylactic acid and its copolymers promote lactate dehydrogenase A expression in dermal papilla cells 1. Grouping and administration: The grouping and administration concentration were the same as in Example 2.

[0144] 2. Cell protein extraction and Western blotting analysis steps: 1) After 16 h of cell pretreatment, cells were lysed using a cell lysis buffer containing protease and phosphatase inhibitors.

[0145] 2) Centrifuge at 12000 rcf for 25 min at 4℃, and determine protein concentration using Coomassie Brilliant Blue. Separate equal volumes of protein by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transfer to a pre-activated methanol-modified polyvinylidene fluoride (PVDF) membrane. Block the PVDF membrane with 5% skim milk at room temperature to prevent the binding of nonspecific proteins.

[0146] 3) Incubate the membrane with an appropriately diluted primary antibody overnight at 4°C. Rinse the membrane with TBST buffer and incubate with secondary antibody at room temperature for 1 hour.

[0147] 4) Enhanced chemiluminescence solution was used to visualize protein bands, with α-Tubulin as an internal control to control for differences in protein load.

[0148] 3. RNA extraction and Real-time PCR reaction steps: 1) According to 800 μL / 5×10 6 Add Trizol to the volume of cells and lyse the cells by shaking. Add 0.2 ml of chloroform to each 1 ml of Trizol, shake vigorously for 15 seconds, and let stand at room temperature for 2-3 minutes.

[0149] 2) Centrifuge at 12000 rcf at 4℃ for 15 min, and carefully aspirate the uppermost layer. Add isopropanol to the aspirated supernatant at a ratio of 0.5 ml per 1 ml Trizol, and let stand at room temperature for 10 min.

[0150] 3) Centrifuge at 12000 rcf at 4℃ for 10 min, discard the supernatant, and obtain the RNA precipitate. Add at least 1 ml of 75% ethanol / 1 ml of Trizol and vortex.

[0151] 4) Centrifuge at 7500 rcf at 4℃ for 5 min, repeat once, and wash the RNA precipitate. Remove all ethanol and air dry for 5-10 min.

[0152] 5) Dissolve the RNA precipitate in an appropriate amount of nuclease-free water, measure the A260 / A280 ratio, and analyze the purity and concentration of total RNA.

[0153] 6) Transcribe 500 ng of total RNA into cDNA. Quantify gene expression using a real-time quantitative PCR system. CT values ​​are calculated using a 2-1Transcription method. −ΔΔCT The method was used for analysis. The primer sequences (5' ~ 3') are as follows: ldha F: 5'-ACGCAGACAAGGAGCAGTGGAA-3' ldha R: 5'-ATGCTCTCAGCCAAGTCTGCCA-3' Tgfb2 F: 5'-TTGTTGCCCTCCTACAGACTGG-3' Tgfb2 R: 5'-GTAAAGAGGGCGAAGGCAGCAA-3' GAPDH F: 5'-CATCACTGCCACCCAGAAGACTG-3' GAPDH R: 5'-ATGCCAGTGAGCTTCCCGTTCAG-3' 4. Experimental Results: Western blot and qRT-PCR results are as follows: Figure 6 , Figure 7 As shown, the levels of Ldha protein and its expression were both upregulated in groups B-F compared to the control group.

[0154] Based on existing research, increased Ldha protein expression can promote the activation of hair follicle stem cells. Therefore, the above results indicate that polylactic acid and its copolymers can promote hair follicle development and regeneration by increasing the expression of Ldha protein in DPC cells.

[0155] Example 4: Polylactic acid and its copolymers promote the expression and secretion of TGF-β2 in dermal papilla cells. 1. Grouping and administration: The grouping and administration concentration were the same as in Example 2.

[0156] 2. Experimental methods and procedures: Changes in mRNA expression levels were detected by qRT-PCR, following the same experimental procedure as in Example 3.

[0157] ELISA detection of TGF-β2 secretion: 1) Seed 1.5 mL of cell suspension into each well of a 12-well cell culture plate. Pre-culture the plate overnight at 37°C in a 5% CO2 incubator. The culture medium was serum-free and supplemented with insulin-transferrin-selenium to provide the necessary nutrients for the DPC cells.

[0158] 2) Add the microspheres prepared according to the corresponding preparation examples to the wells, and incubate the culture plate in an incubator for 48 h. Collect the cell supernatant sample and centrifuge at 200 g for 5 min.

[0159] 3) After removing the ELISA kit (purchased from Yunclone) from the refrigerator, allow it to equilibrate at room temperature for 20 min. Follow the instructions in the kit manual to perform serial dilutions of the standards, ultimately obtaining six standard concentrations of 1000, 500, 250, 125, 62.5, and 31.25 pg / mL. Add the diluted standards to the pre-coated wells, with the standard dilution buffer serving as the 0 pg / mL concentration, for a total of seven standard concentrations.

[0160] 4) Add 100 μL of the sample or standard of different concentrations to the corresponding well, seal the reaction well with sealing film, and incubate at room temperature for 120 min. Wash the plate 5 times, and pat it dry on thick absorbent paper for the last time.

[0161] 5) Add 100 μL of biotinylated antibody to each well. Seal the wells with sealing film and incubate at room temperature for 60 min. Wash the plate 5 times and pat dry.

[0162] 6) Add 100 μL of horseradish peroxidase-labeled streptavidin to each well, seal the wells with sealing film, and incubate at room temperature in the dark for 20 min. Wash the plate 5 times and pat dry.

[0163] 7) Add 100 μL of TMB solution as a colorimetric reagent per well, seal the wells with a sealing film, and incubate at room temperature in the dark for 20 min until the standard shows a very significant color change.

[0164] 8) Add 50 μL of stop solution per well, mix well, and immediately measure the A450 value using an ELISA reader.

[0165] 3. Experimental Results: See results Figure 8 The ELISA test results were consistent with the qRT-PCR results. After microsphere treatment, the secretion and expression levels of TGF-β2 in DPC cells of groups B to F were significantly increased.

[0166] The above results indicate that administration of polylactic acid and its copolymers can promote the secretion and expression of TGF-β2 in DPC cells.

[0167] Example 5: Polylactic acid and its copolymers promote hair follicle growth and damage repair in mice. 1. Grouping and administration:

[0168] Group A (hair follicle damage model group): Each mouse was injected intraperitoneally with 200 µL of DTH solution (DTH powder dissolved in corn oil to prepare a 5 mg / mL clear solution, DTH powder purchased from Ronghe Pharmaceutical) for 3 consecutive days, and the hair on the back was removed (the area was 5 cm × 5 cm). Then, DTH solution was injected daily for 28 days. Group B (PLGA solid microsphere group): Each mouse was injected intraperitoneally with 200 µL of DTH solution for 3 consecutive days, the hair on the back was removed, and 100 µL of PLGA solid microspheres (prepared as Example 14 microspheres suspended in physiological saline for injection, concentration 30 mg / mL) were injected subcutaneously. DTH solution was then injected daily for 28 days. Group C (mPEG-PLLA porous microspheres): Each mouse was intraperitoneally injected with 200 µL of DTH solution for 3 consecutive days, the hair on the back was removed, and 100 µL of mPEG-PLLA porous microspheres (prepared as Example 10 microspheres suspended in physiological saline for injection, concentration 30 mg / mL) were subcutaneously injected. DTH solution was then injected daily for 28 days. Group D (PLGA porous microsphere group): Each mouse was injected intraperitoneally with 200 µL of DTH solution for 3 consecutive days, the hair on the back was removed, and 100 µL of PLGA porous microspheres (prepared as Example 6 microspheres suspended in physiological saline for injection, concentration 30 mg / mL) were injected subcutaneously. DTH solution was then injected daily for 28 days. Group E (PLLA porous microsphere group): Each mouse was injected intraperitoneally with 200 µL of DTH solution for 3 consecutive days, the hair on the back was removed, and 100 µL of PLLA porous microspheres (prepared as Example 1 microspheres suspended in physiological saline for injection, concentration 30 mg / mL) were injected subcutaneously. DTH solution was then injected daily for 28 days.

[0169] 2. Experimental methods and procedures: Preparation of paraffin sections: 1) Sample collection: After the mouse is euthanized, the target skin tissue (approximately 0.5cm x 0.5cm in area and no more than 0.2cm in thickness) is gently cut along the skin edge with dissecting scissors. 2) Cleansing: Gently rinse 3 times with saline solution to remove impurities from the skin surface; 3) Fixation: Add 4% paraformaldehyde fixative to the cleaned skin tissue and fix at room temperature for 24 hours; 4) Dehydration: After fixation, the skin tissue is rinsed with distilled water for 5 minutes (to remove residual fixative), then transferred to a gradient of ethanol for dehydration, with each step soaking at room temperature for 60 minutes. 5) Transparent: Immerse in a 1:1 mixture of ethanol and xylene for 10 minutes; immerse in xylene I for 15 minutes; immerse in xylene II for 15 minutes. 6) Paraffin infusion: The transparent skin tissue is transferred into paraffin in a 60℃ incubator and infused in three stages. 7) Embedding: Pour the melted paraffin into the embedding box. After a thin layer of paraffin solidifies on the surface, gently place the skin tissue flat into the box using ophthalmic forceps. 8) Slicing: Fix the embedded block on the sample holder of the microtome, adjust the slice thickness to 4-5μm, and slice gently and at a constant speed.

[0170] HE staining: 1) Dewaxing: Place the dried slices in xylene I (10 min) → xylene II (10 min) to completely remove the paraffin wax; 2) Rehydration: Sequentially transfer 100% ethanol I (5 min) → 100% ethanol II (5 min) → 95% ethanol (5 min) → 85% ethanol (5 min) → 75% ethanol (5 min), and finally rinse with distilled water for 3 min (2 times) to complete rehydration; 3) Hematoxylin staining: Place in hematoxylin staining solution and stain at room temperature for 5-8 minutes; 4) Washing: Rinse with running tap water for 10 minutes to remove residual dye; 5) Differentiation: Immerse in 1% hydrochloric acid ethanol differentiation solution for 1-3 seconds; 6) Blueing: Immediately transfer to 0.5% ammonia blueing solution, soak for 10-20 seconds until the slices turn blue, then rinse with tap water for 5 minutes; 7) Eosin counterstaining: Place in eosin staining solution and stain at room temperature for 30 seconds to 1 minute; 8) Dehydration and clearing: Sequentially transfer 95% ethanol I (1 min) → 95% ethanol II (1 min) → 100% ethanol I (2 min) → 100% ethanol II (2 min) → xylene I (5 min) → xylene II (5 min) to quickly dehydrate and clear the product, avoiding eosin discoloration; 9) Mounting: Place 1 drop of neutral resin in the center of the slide, gently cover with a coverslip and press lightly to ensure the resin evenly covers the slide.

[0171] Microscopic examination was performed on the tissue sections to observe the morphology of hair follicles and to count the number of hair follicles and their average diameter.

[0172] 3. Experimental Results: The results are as follows Figure 9 The results showed that the trends in the number of hair follicles and the average diameter of hair follicles in mice were consistent with the aforementioned cell experiments, with PLGA porous microspheres showing the best performance. Compared to the model group, each microsphere treatment group exhibited reduced hair follicle damage, a greater number of hair follicles, and a larger average hair follicle diameter.

[0173] The above results indicate that administration of polylactic acid and its copolymers can effectively promote hair follicle growth and damage repair.

[0174] It is worth noting that only representative data (groups B to F) are shown here. The microparticles / microspheres prepared in Examples 1 to 16 all showed varying degrees of effects in promoting the proliferation of dermal papilla cells (DPCs), upregulating lactate dehydrogenase A expression in DPCs, promoting TGF-β2 secretion in DPCs, and promoting hair follicle growth. The overall trend is consistent with the above results.

[0175] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Use of polylactic acid and / or polylactic acid copolymers in the preparation of articles that promote hair follicle growth and / or repair hair follicle damage.

2. Use of polylactic acid and / or polylactic acid copolymers in the preparation of articles that promote the proliferation of dermal papilla cells.

3. Use of polylactic acid and / or polylactic acid copolymers in the preparation of products that upregulate lactate dehydrogenase A expression in dermal papilla cells.

4. Use of polylactic acid and / or polylactic acid copolymers in the preparation of products that promote the secretion of TGF-β2 from dermal papilla cells.

5. The use according to any one of claims 1-4, characterized in that: The polylactic acid and / or polylactic acid copolymer is prepared as microparticles; preferably, the microparticles are microspheres.

6. The use according to claim 5, characterized in that: The microspheres are porous microspheres; preferably, the porosity of the porous microspheres is 10% to 95%.

7. The use according to claim 5, characterized in that: The particle size is 50 nm to 100 μm; preferably, the particle size is 10 μm to 50 μm; more preferably, the particle size is 15 μm to 35 μm.

8. The use according to any one of claims 1-4, characterized in that: The molecular weight of the polylactic acid and / or polylactic acid copolymer is 400 Da to 50 kDa.

9. The use according to any one of claims 1-4, wherein the polylactic acid comprises one or more of L-polylactic acid and racemic polylactic acid; preferably, the polylactic acid is L-polylactic acid.

10. The use according to any one of claims 1-4, characterized in that: The polylactic acid copolymer includes one or more of polylactic acid-glycolic acid copolymer, polylactic acid-polyethylene glycol copolymer, polyethylene glycol-polylactic acid-glycolic acid copolymer, and lactide-caprolactone copolymer.

11. The use according to any one of claims 1-4, characterized in that: The content of L-lactic acid in the polylactic acid and / or polylactic acid copolymer is at least 20%.

12. The use according to any one of claims 1-4, characterized in that: The polylactic acid and / or polylactic acid copolymer has a content of 0.1% to 80% when administered, more preferably 0.5%, 1%, or 2%.

13. The use according to any one of claims 1-4, characterized in that: The product is one or more of the following: an injection, a microsphere formulation, or a gel.

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