Use of polylactic acid and its copolymers in promoting bone and cartilage tissue growth.

Abstract

This invention provides the use of polylactic acid and its copolymers or their microspheres in the preparation of agents that promote the growth of bone and cartilage tissue, wherein the molecular weight of the polylactic acid and its copolymers is 400 Da to 300 kDa. This invention is the first to discover that polylactic acid and its copolymers or their microspheres have the effect of promoting the growth of bone and cartilage tissue, and provides a novel approach for promoting the growth of bone and cartilage tissue.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the priority of Chinese Patent Application No. 202310853821.9, titled "Use of Polylactic Acid and Its Copolymers in Promoting the Growth of Cartilage Tissue", filed on July 12, 2023, and the entire content of the application is incorporated herein by reference.

[0002] Technical Field The present invention relates to the technical field of polymer medical materials, and particularly to the use of polylactic acid and its copolymers in promoting the growth of bone tissue and cartilage tissue.

Background Art

[0003] The molecular formula of polylactic acid (PLA) is -[OCH(CH3)CO]-, and it is mainly prepared by lactic acid polymerization or ring - opening polymerization of lactide. Its structural formula is,

Chemical Formula

[0004] PLA and its copolymers not only have excellent mechanical strength and chemical stability, but also have good biocompatibility and biodegradability. In recent years, numerous studies have been conducted on its applications in the biomedical field at home and abroad. It is widely used in the fields of surgical sutures, bone repair materials, drug - controlled release systems, and tissue - function scaffolds (artificial bones, artificial skins, etc.).

[0005] Among them, poly - L - lactic acid (trade name SculptraTM, Poly - L - lactic acid, PLLA) contains biodegradable synthetic polymer poly - L - lactic acid microparticles with good biocompatibility. It received approval from the US FDA in 2004 and is used for filling areas of the face of AIDS patients where the skin has sunken or sagged due to lipoatrophy. In 2009, the US FDA officially approved the use of poly - L - lactic acid fillers for improving nasolabial folds.

[0006] However, there are no prior art reports on whether polylactic acid, its copolymers, or their microspheres can be used as polymeric agents to promote the growth of bone and cartilage tissue. [Overview of the project]

[0007] In contrast to the shortcomings of the prior art, the present invention provides the use of polylactic acid and its copolymers or its microspheres in the preparation of agents that promote the growth of bone and cartilage tissue, thereby providing a novel approach to promoting the growth of bone and cartilage tissue.

[0008] The present invention provides the use of polylactic acid and its copolymers, polylactic acid microspheres, and polylactic acid copolymer microspheres in the preparation of agents that promote the growth of bone tissue and cartilage tissue, wherein the molecular weight of the polylactic acid and its copolymers is 400 Da to 300 kDa, preferably 5000 Da to 100 kDa, for example 5000 Da to 20 kDa and 15000 Da.

[0009] According to embodiments of the present invention, the polylactic acid includes levorotatory polylactic acid, dextrorotatory polylactic acid, and racemic polylactic acid, and is preferably levorotatory polylactic acid.

[0010] According to embodiments of the present invention, the polylactic acid copolymer comprises one or more of polylactic acid glycolic acid copolymer, polylactic acid polyethylene glycol copolymer, polyethylene glycol polylactic acid glycolic acid copolymer, polylactic acid-chitosan copolymer, and lactidocaprolactone copolymer, and preferably the polylactic acid copolymer is polylactic acid glycolic acid copolymer.

[0011] According to embodiments of the present invention, a method for preparing polylactic acid microspheres and polylactic acid copolymer microspheres includes the following steps. 1) Mix polylactic acid or polylactic acid copolymer with a solvent to obtain a polylactic acid solution or a polylactic acid copolymer solution. Preferably, the solvent is one of dichloromethane, chloroform, ethyl acetate, acetone, or toluene, or a mixture of two or more of these. 2) A polylactic acid solution or polylactic acid copolymer solution is mixed with an aqueous polyvinyl alcohol solution, emulsified at high speed, stirred to remove the solvent, and freeze-dried to obtain polylactic acid microspheres.

[0012] According to embodiments of the present invention, in step 1), the mass-volume ratio of polylactic acid or polylactic acid copolymer to solvent is 1g:5mL to 1g:30mL, preferably 1g:10mL to 1g:20mL, for example 1g:15mL. And / or, in step 2), the mass concentration of the aqueous polyvinyl alcohol solution is 0.05% to 5.0%, preferably 0.1% to 2.0%, for example 0.5%. And / or, in step 2), the volume ratio of the polylactic acid solution or polylactic acid copolymer solution to the polyvinyl alcohol aqueous solution is 1:5 to 1:30, preferably 1:10 to 1:20, for example 1:13 or 1:15. And / or, in step 2), the rotational speed for high-speed emulsification is 1000 rpm / min to 5000 rpm / min, for example, 3000 rpm / min. And / or, in step 2), the emulsification time is 5 to 20 minutes, for example, 10 minutes.

[0013] According to embodiments of the present invention, at the time of administration, the mass concentration of polylactic acid and its copolymer, polylactic acid microspheres, and polylactic acid copolymer microspheres is 1% to 60%, preferably 5% to 50%, for example 10%, 20%, 30%, or 40%.

[0014] For example, prepare a solution of the above mass concentration.

[0015] According to embodiments of the present invention, the agent further optionally comprises auxiliary materials, Preferably, the auxiliary material comprises at least one of a stabilizer, filler, binder, surfactant, and lubricant. Preferably, the surfactant is selected from one or more of polyethylene glycol, sodium dodecyl sulfate, Zween, and Span. Preferably, the stabilizer is selected from one or more of carboxymethylcellulose and mannitol. Preferably, the filler is selected from one or more of lactose, mannitol, cyclodextrin, and sorbitol. Preferably, the binder is selected from one or more of hydroxypropylcellulose, methylcellulose, sodium hyaluronate, collagen, and polyvinylpyrrolidone. Preferably, the lubricant is selected from one or more of magnesium stearate, calcium stearate, and stearic acid.

[0016] According to embodiments of the present invention, the agent may optionally be used in combination with other active ingredients that have the effect of promoting the growth of bone tissue and cartilage tissue. Preferably, the other active ingredients are selected from one or more of the following: collagen, gelatin, osteocalcin (BGP), bone morphogenetic protein (BMP), basic fibroblast growth factor (bFGF), insulin growth factor (IGF), transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), concentrated growth factor (CGF), and platelet-rich fibrin (PRF).

[0017] According to embodiments of the present invention, the administration method of the drug includes, but is not limited to, intravenous injection, in situ injection, intramuscular injection, subcutaneous injection, oral administration, topical application, etc.

[0018] According to an embodiment of the present invention, the dosage form of the drug is an injection, tablet, granule, capsule, oral liquid, ointment, etc., and preferably an injection.

[0019] Compared with the prior art, the present invention has the following beneficial effects.

[0020] The present invention first discovers that polylactic acid and its copolymers, polylactic acid microspheres, and polylactic acid copolymer microspheres have the effect of promoting the growth of bone tissue and cartilage tissue, and provides a new approach for promoting the growth of bone tissue and cartilage tissue.

[0021] The accompanying drawings described herein are for providing a further understanding of the present invention and constitute a part of this application. The schematic embodiments and descriptions of the present invention are used to interpret the present invention and do not unduly limit the present invention.

Brief Description of the Drawings

[0022] [Figure 1] It is a scanning electron microscope (SEM) photograph of left-handed polylactic acid (PLLA) microspheres in Example 1. [Figure 2] It is a scanning electron microscope (SEM) photograph of poly(lactic-co-glycolic acid) (PLGA) microspheres in Example 2. [Figure 3] It is a diagram of the influence result of the PLLA microsphere solution on the proliferation behavior of osteoblasts. <00​​​​​​​​​​​​​

[0023] The technical solutions of the present invention will be described in more detail below with reference to specific examples.

[0024] The following examples are for illustrative purposes only and should not be interpreted as limiting the scope of protection of the present invention. Any technology realized based on the above-described aspects of the present invention falls within the scope of protection of the present invention.

[0025] Unless otherwise specified, the raw materials and reagents used in the following examples are either commercially available or can be prepared by known methods. [Examples]

[0026] Example 1: Preparation and Characterization of Levator-Rotator Polylactic Acid Microspheres 9 g of levorotatory polylactic acid (PLLA) (molecular weight 15000 g / mol) was dissolved in 135 mL of dichloromethane. The polymer solution was then added to 1800 mL of a 0.5% polyvinyl alcohol aqueous solution, emulsified at 3000 rpm / min for 10 minutes, and then stirred at 500 rpm for 3 hours to remove the dichloromethane. The mixture was then freeze-dried to obtain levorotatory polylactic acid microspheres.

[0027] Levorotatory polylactic acid microspheres prepared using a scanning electron microscope were observed and calculated to obtain their particle size. The scanning electron microscope (SEM) images of the levorotatory polylactic acid microspheres are shown in Figure 1, and the particle size range of the microspheres was 3.72 to 33.12 μm.

[0028] Example 2: Preparation and Characterization of Polylactic Acid-Glycolic Acid Copolymer Microspheres 9 g of polylactic acid-glycolic acid copolymer (PLGA) (molecular weight 15000 g / mol) was dissolved in 135 mL of dichloromethane. Then, the polymer solution was added to 1800 mL of a 0.5% polyvinyl alcohol aqueous solution, emulsified at 3000 rpm / min for 10 minutes, and then stirred at 500 rpm for 3 hours to remove the dichloromethane. The mixture was then freeze-dried to obtain polylactic acid-glycolic acid copolymer microspheres.

[0029] The particle size of the polylactic acid-glycolic acid copolymer microspheres prepared using a scanning electron microscope was obtained by observation and calculation. The scanning electron microscope (SEM) images of the PLGA microspheres are shown in Figure 2, and the particle size range of the microspheres was 2.94 to 36.92 μm.

[0030] Example 3: Experiment on the effect of levorotatory polylactic acid (PLLA) microsphere solution on osteoblast proliferation. The effect of the PLLA microsphere solution in Example 1 on the proliferation of mouse embryonic osteoblasts (MC3T3) was measured using the MTT method.

[0031] PBS buffer was used as a blank control group, and PLLA microsphere solutions with mass concentrations of 10%, 20%, and 30% were prepared using PBS buffer and used as experimental groups. First, the samples from each group were added to a 96-well plate, with 100 μL in each well, and five parallel wells were provided for each sample. Next, 100 μL of a solution with a concentration of 1 × 10⁶ was added to each sample well. 4 The cells were placed in a cell incubator after adding a further MC3T3 cell suspension at a concentration of / mL. After incubation in the dark for 1, 3, 5, and 7 days, 20 μL of MTT solution was added to each well, and the cells were incubated in the dark for 4 hours. The supernatant from each well was then removed and dissolved with 150 μL of dimethyl sulfoxide until a clear solution was obtained. The readings at 570 nm were measured using a microplate reader, and the effects of different concentrations of PLLA microsphere solution on the proliferation behavior of MC3T3 cells were finally calculated.

[0032] The results are shown in Figure 3. Compared to the blank control group, all different concentrations of PLLA microsphere solution were able to promote osteoblast proliferation, and the number of osteoblasts gradually increased with increasing PLLA microsphere concentration. This indicates that PLLA microsphere solution has a significant effect in promoting osteoblast proliferation.

[0033] Example 4: Experiment on the effect of levorotatory polylactic acid (PLLA) microsphere solution on bone tissue growth. 1. Construction of an osteoporosis model: Forty non-pregnant female SD rats, each weighing approximately 200g, were selected. Both ovaries were removed by castration surgery, and the rats were kept for three months post-operatively.

[0034] 2. Microsphere transplantation and detection of bone formation status in vivo: (1) Specimen preparation After anesthetizing the rats, they were placed prone on the operating table. The greater trochanters of both femoral bones were incised and exposed, and a bone defect 2.5 mm in diameter and 3 mm deep was created using an electric drill, running from the greater trochanter towards the lesser trochanter. Forty castrated rats were randomly divided into five groups of eight rats each. The groups were as follows: control group (sham surgery performed), buffer group (PBS buffer injected into the bone defect area between the femoral trochanters), low-concentration PLLA microsphere group (5% microspheres / PBS buffer), medium-concentration PLLA microsphere group (10% microspheres / PBS buffer), and high-concentration PLLA microsphere group (20% microspheres / PBS buffer). 30 μL of each of the above samples was injected into the bilateral defect area of ​​the rats, and the muscle and skin were sutured in layers. Three months post-surgery, eight rats from each group were sacrificed, complete femurs were removed, soft tissue was decomposed, and the bones were wrapped in saline gauze and stored at -20°C. The PLLA microspheres used were those prepared in Example 1.

[0035] (2) Measurement of bone mineral density in the intertrochanteric region of the femur Femur specimens were scanned using an X-ray bone mineral density meter to measure bone mineral content (BMC) and bone mineral density (BMD) in the intertrochanteric region of the femur. As can be seen from the data in Table 1, at 3 months post-surgery, the bone mineral content and bone mineral density in the microsphere sample group were both higher than those in the control group and the buffer solution group, and the values ​​of bone mineral content and bone mineral density gradually increased with increasing microsphere concentration (the comparative differences in bone mineral content and bone mineral density in the intertrochanteric region of the femur were all statistically significant).

[0036] [Table 1]

[0037] (3) Observation of trabecular structure in the intertrochanteric region of the femur using Micro CT The femur was cut from the lower edge of the lesser trochanter, and the proximal portion was placed in a sample container along its long axis. The sample was scanned under conditions of 80kV voltage and 6.8μm resolution to measure bone morphometric indices in the intertrochanteric region of the femur. Among these, 1) Percentage of trabecular area (%Tb.Ar): This is the percentage of trabecular area to total bone tissue area and reflects the amount of trabecular bone. 2) Trabecular thickness (Tb.Th): Used to describe the morphology of the trabecular structure. Under conditions where the quantity is constant, a larger thickness indicates a greater amount of trabecular bone. 3) Trabecular spacing (Tb.Sp): Used to describe the average distance between trabecular bones. A larger spacing indicates wider trabecular spacing and sparser bone quality.

[0038] As can be seen from the data in Table 2, at 3 months post-surgery, the trabecular bone thickness values ​​in the microsphere sample group were significantly higher than those in the control and buffer groups, and the trabecular bone thickness gradually increased with increasing microsphere concentration (all differences between groups were statistically significant). Furthermore, the trabecular bone area percentage in the 20% microsphere sample group was significantly higher than that in the control and buffer groups, but the difference was not statistically significant compared to the 5% and 10% microsphere groups. At 3 months post-surgery, the trabecular bone spacing values ​​in the microsphere sample group were lower than those in the control and buffer groups, and the trabecular bone spacing gradually decreased with increasing microsphere concentration (all differences between groups were statistically significant).

[0039] [Table 2]

[0040] Example 5: Experiment on the effects of polylactic acid-glycolic acid copolymer PLGA microsphere solution on chondrocytes. 1. Extraction and culture of cartilage cells Chondrocytes were collected from the cartilage tissue of both ears of 3-week-old New Zealand white rabbits, cultured for about a week, and passed through once. In this invention, third-generation chondrocytes were used for transplantation of microsphere material.

[0041] 2. Testing the effect of PLGA microsphere solution on chondrocyte proliferation using the MTT method. PBS buffer was used as a blank control group, and PLLA microsphere solutions with mass concentrations of 10%, 20%, and 40% were prepared using PBS buffer and used as experimental groups. First, samples from each group were added to a 96-well plate, with 100 μL of microsphere solution in each well, and five parallel wells were provided for each sample. Next, 100 μL of a solution with a concentration of 1 × 10⁶ was added to each sample well. 4After adding a cell suspension of / mL, the cells were placed in a cell incubator. After incubation in the dark for 2, 4, 6, and 8 days, 20 μL of MTT solution was added to each well, and after incubation in the dark for 4 hours, the supernatant from each well was removed and then dissolved with 150 μL of dimethyl sulfoxide until a clear solution was obtained. The reading at 570 nm was measured using a microplate reader, and the proliferation results of chondrocytes in the microsphere samples of different concentrations were finally calculated. The polylactic acid-glycolic acid copolymer microspheres were prepared in Example 2.

[0042] The test results are shown in Figure 4. Compared to the blank control group, all PLGA microsphere solutions at different concentrations were able to promote chondrocyte proliferation. Furthermore, the number of chondrocytes in the sample group gradually increased with increasing culture time and microsphere concentration, indicating that PLGA microspheres can promote chondrocyte proliferation.

[0043] Example 6: Effect of polylactic acid-glycolic acid copolymer PLGA microsphere solution on cartilage tissue growth 1. Isolation of cartilage cells Under sterile conditions, a piece of pig ear cartilage measuring approximately 2 cm x 2 cm was cut out. After separating the skin and subcutaneous tissue under sterile conditions, the perichondrium was separated, measuring approximately 1 mm. 3 The cartilage was cut into blocks and digested with 0.25% trypsin for 40 minutes, then 5 times the volume of 0.2% collagenase was added and digestion continued for 16-24 hours. After filtering the cells through a cell filter, the precipitate was collected by repeated centrifugation, and finally cultured in a constant temperature incubator to obtain P0 generation pig ear chondrocytes.

[0044] 2. Mixture of cartilage cells and PLGA microspheres PLGA microsphere solutions at mass concentrations of 10% and 40%, respectively, were prepared using PBS buffer and used as experimental groups. P2 generation porcine ear chondrocytes were selected, and the density was 1 × 10⁶. 8A cell suspension was prepared at a concentration of / mL. An appropriate amount of cell suspension, PLGA microsphere solution, and culture medium were added together to a 6-well plate and incubated in a constant-temperature incubator. The specific groupings are shown in Table 3.

[0045] [Table 3]

[0046] 3. Real-time PCR After culturing cell microspheres in vitro for 4 weeks, real-time PCR detection of cartilage-related gene expression was performed, and the expression levels of the internal standard gene GAPDH in each group were statistically analyzed.

[0047] 1) Reaction system: 0.3 μl each of upstream and downstream primers, 5 μl of SYBR, 0.4 μl of template, 4 μl of ddH2O. 2) Reaction program: 95°C for 1 minute, 95°C for 5 seconds, 60°C for 1 minute, 40 cycles. 3) Perform quantitative analysis using GAPDH levels in each group as an internal standard. 4) The primer sequences are shown in Table 4.

[0048] [Table 4]

[0049] Figure 5 shows the results of the test of relative expression levels of cartilage-related genes after in vitro culture of microsphere-chondrocyte complexes. As the results show, PLGA microspheres contribute to cartilage generation. Compared to low-concentration microsphere samples, the expression levels of cartilage-related genes were significantly increased in high-concentration microsphere samples, and the difference was statistically significant.

[0050] Example 7: Effect of polylactic acid-glycolic acid copolymer PLGA microsphere solution on cartilage tissue growth 1. In vivo injection of microsphere solution Three healthy, male, 2-month-old miniature pigs weighing 8-10 kilograms were used. After anesthesia and disinfection, subcutaneous cavities were isolated in the pigs according to a sterile procedure. A PBS buffer solution was used as the control group, and 1 mL each of PLGA microsphere solutions prepared in PBS buffer solution with mass concentrations of 20% and 60%, respectively, were used as the sample group. The control and sample groups were injected subcutaneously into both sides of the pig's back, sutured, and marked. Two weeks after injection, the material was removed and placed in formalin for histological staining.

[0051] 2. Histological staining (1) Preparation of tissue sections 1) Trim the tissue fixed in 10% formalin into tissue pieces of appropriate size. 2) Gradient ethanol dehydration: 30 minutes with 70% ethanol, 30 minutes with 80% ethanol, 30 minutes with 90% ethanol, 30 minutes with 95% ethanol I, 30 minutes with 95% ethanol II, 40 minutes with anhydrous ethanol I, 40 minutes with anhydrous ethanol II, 40 minutes with anhydrous ethanol:xylene (1:1), and 8 minutes twice with xylene. 3) After removing from xylene, rapidly add paraffin:xylene (1:1) for 40 minutes, add soft wax with a melting point of 50-52°C for 40 minutes, and add hard wax with a melting point of 60-62°C for 40 minutes. 4) Embed the tissue in hard wax and leave it at room temperature overnight. 5) The thickness of the tissue sections obtained using a tissue slicer is 5-8 μm. 6) Wash with distilled water at 45°C. 7) Dry at 50°C for 1.5 to 2 hours to prepare for use.

[0052] 3. Immunohistochemical staining of type II collagen 1) Allow the frozen sections to recover at room temperature for 1 hour, then fix them in 4% paraformaldehyde for 15 minutes. Wash them by immersion in PBS three times for 3 minutes each time. 2) Perform antigen recovery according to the steps of the pepsin antigen recovery kit, add hydrogen peroxide methanol solution dropwise, incubate at room temperature for 10 minutes, and wash three times with PBS for 3 minutes each time. 3) Incubate with pepsin for 30 minutes, then wash three times with PBS for 3 minutes each time. 4) The immunohistochemistry kit was followed, and the endogenous enzyme was blocked by blocking with a 3% hydrogen peroxide solution at room temperature. 5) Wash three times with PBS for 3 minutes each time. 6) Block nonspecific antigens with 10% goat serum for 10 minutes. 7) Dilute the primary antibody with PBS according to the instructions, discard the goat serum, add the primary antibody dropwise, and incubate overnight at 4°C. 8) Allow to equilibrate at room temperature for 1-2 hours. 9) Discard the primary antibody and wash three times with PBS for 3 minutes each time. 10) Add the secondary antibody and wash at 37°C for 30 minutes. 11) Wash three times with PBS for 3 minutes each time. 12) Prepare the DAB color-developing solution according to the instructions, drop the solution onto the microscope, and stop the color development after a positive result appears. 13) Stain with hematoxylin for 2 minutes, then rinse with water. 14) Immerse in tap water for 10 minutes. 15) Dehydrate with anhydrous ethanol for 5 minutes. 16) Clear with xylene I for 5 minutes, clear with xylene II for 5 minutes, and then encapsulate with a neutral resin.

[0053] The immunohistochemical results are shown in Figure 6. As the results show, two weeks after in vivo injection, all PLGA microsphere solution sample groups showed significant positive expression of type II collagen compared to the PBS buffer solution group, and statistical results indicate that the amount of type II collagen expressed in the high-concentration microsphere solution was significantly higher than that in the low-concentration microsphere solution. As the results show, PLGA microsphere solution can promote cartilage growth.

[0054] Finally, it should be noted that the above embodiments are used solely to illustrate, and not to limit, the technical solutions of the present invention. While the present invention has been described in detail with reference to preferred embodiments, those skilled in the art will understand that the technical solutions of the present invention can be modified or replaced with equivalents without departing from the object and scope of the technical solutions, and such modifications should be included within the scope of the claims of the present invention.

Claims

1. The use of polylactic acid and its copolymers, polylactic acid microspheres, and polylactic acid copolymer microspheres in the preparation of agents that promote the growth of bone tissue and cartilage tissue, wherein the molecular weight of the polylactic acid and its copolymers is 400 Da to 300 kDa. Preferably, the molecular weight of the polylactic acid and its copolymer is 5000 Da to 100 kDa, for example, 5000 Da to 20 kDa or 15000 Da.

2. The use according to claim 1, characterized in that the polylactic acid includes levorotatory polylactic acid, dextrorotatory polylactic acid, and racemic polylactic acid, and is preferably levorotatory polylactic acid.

3. The polylactic acid copolymer comprises one or more of the following: polylactic acid glycolic acid copolymer, polylactic acid polyethylene glycol copolymer, polyethylene glycol polylactic acid glycolic acid copolymer, polylactic acid-chitosan copolymer, and lactidocaprolactone copolymer. Preferably, the use according to claim 1 is characterized in that the polylactic acid copolymer is a polylactic acid glycolic acid copolymer.

4. Methods for preparing polylactic acid microspheres and polylactic acid copolymer microspheres are as follows: Polylactic acid or polylactic acid copolymer is mixed with a solvent to obtain a polylactic acid solution or a polylactic acid copolymer solution. Preferably, the solvent is one of dichloromethane, chloroform, ethyl acetate, acetone, or toluene, or a mixture of two or more of these, in step 1) The use according to any one of claims 1 to 3, characterized by comprising step 2) mixing a polylactic acid solution or a polylactic acid copolymer solution with an aqueous solution of polyvinyl alcohol, emulsifying at high speed, stirring to remove the solvent, and freeze-drying to obtain polylactic acid microspheres.

5. In step 1), the mass-volume ratio of polylactic acid or polylactic acid copolymer to solvent is 1 g:5 mL to 1 g:30 mL, preferably 1 g:10 mL to 1 g:20 mL, for example 1 g:15 mL. And / or, in step 2), the mass concentration of the aqueous polyvinyl alcohol solution is 0.05% to 5.0%, preferably 0.1% to 2.0%, for example 0.5%. And / or, in step 2), the volume ratio of the polylactic acid solution or polylactic acid copolymer solution to the polyvinyl alcohol aqueous solution is 1:5 to 1:30, preferably 1:10 to 1:20, for example 1:13 or 1:

15. And / or, in step 2), the rotational speed for high-speed emulsification is 1000 rpm / min to 5000 rpm / min, for example, 3000 rpm / min. The use according to claim 4, characterized in that, in step 2), the emulsification time is 5 to 20 minutes, for example, 10 minutes.

6. The use according to any one of claims 1 to 5, characterized in that, at the time of administration, the mass concentration of polylactic acid and its copolymer, polylactic acid microspheres, and polylactic acid copolymer microspheres is 1% to 80%, preferably 5% to 50%, for example 10%, 20%, 30%, or 40%.

7. The aforementioned drug may optionally further contain auxiliary materials. Preferably, the auxiliary material includes at least one of a stabilizer, filler, binder, surfactant, and lubricant. Preferably, the surfactant is selected from one or more of polyethylene glycol, sodium dodecyl sulfate, Zween, and Span. Preferably, the stabilizer is selected from one or more of carboxymethylcellulose and mannitol. Preferably, the filler is selected from one or more of lactose, mannitol, cyclodextrin, and sorbitol. Preferably, the binder is selected from one or more of hydroxypropylcellulose, methylcellulose, sodium hyaluronate, collagen, and polyvinylpyrrolidone. Preferably, the lubricant is selected from one or more of magnesium stearate, calcium stearate, and stearic acid, as described in any one of claims 1 to 6.

8. The aforementioned drug may be optionally used in combination with other active ingredients that have the effect of promoting the growth of bone tissue and cartilage tissue. Preferably, the other active ingredients are collagen, gelatin, osteocalcin (Bone Gla Protein, BGP), bone morphogenetic protein (BMP), basic fibroblast growth factor (bFGF), insulin growth factor (IGF), transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), CGF-containing fibrin (Concentrated Growth Factor, The use according to any one of claims 1 to 7, characterized in that one or more of the following are selected: CGF and Platelet-Rich Fibrin (PRF).

9. The use according to any one of claims 1 to 8, characterized in that the form of administration of the drug includes, but is not limited to, intravenous injection, in situ injection, intramuscular injection, subcutaneous injection, oral administration, topical application, etc.

10. The use according to any one of claims 1 to 8, characterized in that the dosage form of the drug is an injection, tablet, granule, capsule, oral liquid, ointment, etc., and preferably an injection.

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