Application of polylactic acid and copolymer thereof in promoting growth of bone tissues and cartilage tissues

Abstract

The invention provides application of polylactic acid and a copolymer thereof or microspheres thereof in preparation of medicines for promoting growth of bone tissues and cartilage tissues. The molecular weight of the polylactic acid and the copolymer thereof is 400 Da to 300 kDa. The invention firstly discovers that the polylactic acid and the copolymer thereof or the microspheres thereof have the effect of promoting the growth of the bone tissue and the cartilage tissue, and provides a new way for promoting the growth of the bone tissue and the cartilage tissue.

Description

Technical Field

[0001] The invention relates to the technical field of polymer medical materials, and in particular to the application of polylactic acid and its copolymers in promoting the growth of bone tissue and cartilage tissue. Background Art

[0002] The molecular formula of polylactic acid (PLA) is -[OCH(CH3)CO]-, which is mainly prepared by lactic acid polymerization or lactide ring-opening polymerization. The structural formula is

[0003]

[0004] PLA and its copolymers not only have excellent mechanical strength and chemical stability, but also have good biocompatibility and biodegradability. In recent years, a lot of research has been done on its application in biomedicine at home and abroad. It has been widely used in surgical sutures, bone repair materials, drug controlled sustained release systems, and tissue functional scaffolds (such as artificial bones and artificial skin).

[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. In 2004, it was approved by the US FDA for filling the sagging skin areas caused by facial fat atrophy in AIDS patients. In 2009, the US FDA officially approved poly-L-lactic acid fillers for improving nasolabial folds.

[0006] However, there is no report in the prior art as to whether polylactic acid and its copolymers or microspheres thereof can be used as polymer drugs to promote the growth of bone tissue and cartilage tissue. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides the use of polylactic acid and its copolymers or their microspheres in the preparation of drugs for promoting the growth of bone tissue and cartilage tissue, which provides a new approach to promote the growth of bone tissue and cartilage tissue.

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

[0009] According to an embodiment of the present invention, the polylactic acid includes L-polylactic acid, D-polylactic acid, and racemic polylactic acid; preferably, L-polylactic acid.

[0010] According to an embodiment of the present invention, 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, polylactic acid-chitosan copolymer, and lactide-caprolactone copolymer; preferably, the polylactic acid copolymer is a polylactic acid glycolic acid copolymer.

[0011] According to an embodiment of the present invention, the method for preparing polylactic acid microspheres and polylactic acid copolymer microspheres comprises the following steps:

[0012] 1) mixing polylactic acid or a polylactic acid copolymer and a solvent to obtain a polylactic acid solution or a polylactic acid copolymer solution;

[0013] Preferably, the solvent is any one of dichloromethane, chloroform, ethyl acetate, acetone or toluene, or a mixture of any two or more thereof;

[0014] 2) Mixing the polylactic acid solution or the polylactic acid copolymer solution with the polyvinyl alcohol aqueous solution, emulsifying at high speed, stirring to remove the solvent, and freeze-drying to obtain polylactic acid microspheres.

[0015] According to an embodiment of the present invention, in step 1), the mass volume ratio of polylactic acid or polylactic acid copolymer to solvent is 1g:5mL-1g:30mL, preferably 1g:10mL-1g:20mL, for example 1g:15mL;

[0016] and / or, in step 2), the mass concentration of the polyvinyl alcohol aqueous solution is 0.05%-5.0%, preferably 0.1%-2.0%, for example 0.5%;

[0017] And / or, in step 2), the volume ratio of the polylactic acid solution or the polylactic acid copolymer solution to the polyvinyl alcohol aqueous solution is 1:5-1:30, preferably 1:10-1:20, for example 1:13, 1:15;

[0018] And / or, in step 2), the rotation speed of the high-speed emulsification is 1000 rpm / min-5000 rpm / min, for example 3000 rpm / min;

[0019] And / or, in step 2), the emulsification time is 5-20 minutes, for example 10 minutes.

[0020] According to an embodiment of the present invention, when administered, the mass concentration of polylactic acid and its copolymers, polylactic acid microspheres, and polylactic acid copolymer microspheres is 1%-60%, preferably 5%-50%, for example 10%, 20%, 30%, 40%.

[0021] For example, a solution with the above mass concentration is prepared.

[0022] According to an embodiment of the present invention, the drug may optionally further include excipients;

[0023] Preferably, the auxiliary material includes at least one of a stabilizer, a filler, a binder, a surfactant and a lubricant;

[0024] Preferably, the surfactant is selected from one or more of polyethylene glycol, sodium dodecyl sulfate, Tween, and Span;

[0025] Preferably, the stabilizer is selected from one or both of carboxymethyl cellulose and mannitol;

[0026] Preferably, the filler is selected from one or more of lactose, mannitol, cyclodextrin, and sorbitol;

[0027] Preferably, the binder is selected from one or more of hydroxypropyl cellulose, methyl cellulose, sodium hyaluronate, collagen, and polyvinyl pyrrolidone;

[0028] Preferably, the lubricant is selected from one or more of magnesium stearate, calcium stearate and stearic acid.

[0029] According to an embodiment of the present invention, the drug may optionally be further used in combination with other active ingredients, and the other active ingredients have the effect of promoting the growth of bone tissue and cartilage tissue;

[0030] Preferably, the other active ingredients are selected from one or more of collagen, gelatin, osteocalcin (Bone Gla Protein, BGP), bone morphogenetic protein (Bonemorphogenetic protein, BMP), basic fibroblast growth factor (Basic Fibroblast Growth Factor, bFGF), insulin growth factor (Insulin Iike Growth Factor, IGF), transforming growth factor β (Transforming Growth Factor-β, TGF-β), platelet derived growth factor (Plateletderivedgrowth factor, PDGF), concentrated growth factor fibrin (Concentrated GrowthFacter, CGF) and platelet-rich fibrin (Platelet-Rich Fibrin, PRF), etc.

[0031] According to an embodiment 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, application, etc.

[0032] According to an embodiment of the present invention, the dosage form of the drug is injection, tablet, granule, capsule, oral liquid, ointment, etc.; preferably injection.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention discovers for the first time that polylactic acid and its copolymers, polylactic acid microspheres, and polylactic acid copolymer microspheres have the function 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a scanning electron microscope (SEM) image of the left-handed polylactic acid (PLLA) microspheres in Example 1.

[0036] Figure 2 This is a scanning electron microscope (SEM) image of the polylactic acid-co-glycolic acid (PLGA) microspheres in Example 2.

[0037] Figure 3 This is a graph showing the effect of PLLA microsphere solution on the proliferation behavior of osteoblasts.

[0038] Figure 4 This is a graph showing the effect of PLGA microsphere solution on the proliferation behavior of chondrocytes.

[0039] Figure 5 These are the test results of the relative expression levels of cartilage-related genes after in vitro culture of PLGA microsphere solution-cell complex.

[0040] Figure 6 The immunohistochemical results show the effect of PLGA microsphere solution on cartilage tissue growth. DETAILED DESCRIPTION

[0041] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations 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 included in the scope that the present invention is intended to protect.

[0042] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0043] Example 1 Preparation and Characterization of L-polylactic acid Microspheres

[0044] 9 g of L-polylactic acid (PLLA) (molecular weight 15000 g / mol) was dissolved in 135 mL of dichloromethane, and then the above polymer solution was added into 1800 mL of 0.5% polyvinyl alcohol aqueous solution, and after emulsification at 3000 rpm / min for 10 minutes, the dichloromethane was removed by stirring at 500 rpm for 3 hours, and L-polylactic acid microspheres were obtained after freeze-drying.

[0045] The prepared L-polylactic acid microspheres were observed by scanning electron microscopy and the particle size was calculated. The SEM image of the L-polylactic acid microspheres is shown in Figure 1 As shown, the particle size of the microspheres ranges from 3.72 to 33.12 μm.

[0046] Example 2 Preparation and Characterization of Poly(lactic acid-co-glycolic acid) Microspheres

[0047] 9 g of poly(lactic acid-co-glycolic acid) (PLGA) (molecular weight 15000 g / mol) was dissolved in 135 mL of dichloromethane, and then the polymer solution was added into 1800 mL of a 0.5% poly(vinyl alcohol) aqueous solution, and the mixture was emulsified at 3000 rpm / min for 10 minutes and then stirred at 500 rpm for 3 hours to remove dichloromethane, and poly(lactic acid-co-glycolic acid) microspheres were obtained after freeze drying.

[0048] The prepared polylactic acid-co-glycolic acid microspheres were observed by scanning electron microscopy and the particle size was calculated. The scanning electron microscope (SEM) image of the PLGA microspheres is shown in Figure 2 As shown, the particle size of the microspheres ranges from 2.94 to 36.92 μm.

[0049] Example 3 Experiment on the effect of poly (L-lactic acid) (PLLA) microsphere solution on osteoblast proliferation

[0050] The MTT method was used to test the effect of the PLLA microsphere solution in Example 1 on the proliferation of mouse embryonic osteoblasts MC3T3.

[0051] PBS buffer was used as the blank control group, and 10%, 20%, and 30% PLLA microsphere solutions were prepared with PBS buffer as the experimental groups. First, the samples of each group were added to a 96-well plate, 100 μL per well, and 5 parallel wells were set for each sample. Then, 100 μL of 1×10 4 / mL MC3T3 cell suspension was placed in a cell culture incubator. After incubation in the dark for 1, 3, 5 and 7 days, 20μL of MTT solution was added to each well. After incubation in the dark for 4 hours, the supernatant in each well was removed and then dissolved into a transparent solution with 150μL of dimethyl sulfoxide. The reading at 570nm was tested by an ELISA instrument, and the effect of different concentrations of PLLA microsphere solution on the proliferation behavior of MC3T3 cells was finally calculated.

[0052] The results are as follows Figure 3 As shown in the figure, compared with the blank control group, different concentrations of PLLA microsphere solutions can promote the proliferation of osteoblasts; and as the concentration of PLLA microspheres increases, the number of osteoblast proliferation also gradually increases. It can be seen that the PLLA microsphere solution has a significant effect on promoting the proliferation of osteoblasts.

[0053] Example 4 Experimental study on the effect of poly(L-lactic acid) PLLA microsphere solution on bone tissue growth

[0054] 1. Establishment of osteoporosis model:

[0055] Forty 3-month-old non-pregnant female SD rats weighing about 200 g were selected and underwent castration surgery to remove bilateral ovaries. The rats were then fed for 3 months after surgery.

[0056] 2. Implantation of microspheres and detection of in vivo osteogenesis:

[0057] (1) Specimen preparation

[0058] The rats were anesthetized and placed prone on the surgical panel. The bilateral greater trochanters of the femur were cut and exposed, and a bone defect of 2.5 mm in diameter and 3 mm in depth was made from the greater trochanter to the lesser trochanter with an electric drill. 40 castrated rats were randomly divided into 5 groups, 8 in each group. They were: control group (sham operation), buffer group (PBS buffer was injected into the intertrochanteric bone defect area), low-concentration PLLA microsphere group (5% microsphere / PBS buffer), medium-concentration PLLA microsphere group (10% microsphere / PBS buffer), and high-concentration PLLA microsphere group (20% microsphere / PBS buffer). 30 μL of the above sample was injected into the defects on both sides of the rats, and the muscles and skin were sutured in layers. Three months after the operation, 8 rats in each group were killed, the intact femur was taken out, the soft tissue was cleaned, and the saline gauze was wrapped and stored at -20°C. The PLLA microspheres used were prepared in Example 1.

[0059] (2) Bone density measurement of the intertrochanteric region

[0060] The femoral specimens were scanned by X-ray bone densitometer to measure the bone mineral content (BMC) and bone mineral density (BMD) in the intertrochanteric region of the femur. From the data in Table 1, it can be seen that the bone mineral content and bone density of the microsphere sample group were not lower than those of the control group and the buffer solution group 3 months after surgery, and with the increase of microsphere concentration, the bone mineral content and bone density values ​​gradually increased (the comparison of bone mineral content and bone density in the intertrochanteric region of the femur was statistically significant).

[0061] Table 1 Comparison of intertrochanteric bone mineral content and bone density of different samples 3 months after surgery (n = 8)

[0062]

[0063] (3) Micro CT observation of trabecular bone structure in the intertrochanteric region of the femur

[0064] The femur was cut off from the lower edge of the lesser trochanter, and the proximal part was placed in a sample container along the long axis. The femur was scanned at a voltage of 80 kV and a resolution of 6.8 μm to measure the bone morphometric indicators of the intertrochanteric region of the femur. Among them: 1) percentage of trabecular area (% Tb.Ar): the percentage of trabecular area to bone tissue area, reflecting the amount of bone; 2) trabecular thickness (Tb.Th): used to describe the morphology of trabecular structure. Under the condition of a certain amount, the greater the thickness, the more bone mass; 3) trabecular spacing (Tb.Sp): used to describe the average distance between trabeculae. The greater the separation, the greater the trabecular spacing, and the more porous the bone.

[0065] As can be seen from the data in Table 2, the trabecular thickness values ​​of the microsphere sample group 3 months after surgery were significantly higher than those of the control group and the buffer group, and as the concentration of the microspheres increased, the trabecular thickness gradually increased (the differences between the groups were statistically significant). In addition, the percentage of trabecular area in the 20% microsphere sample group was significantly higher than that in the control group and the buffer group, but there was no statistically significant difference compared with the 5% and 10% microsphere groups. The trabecular separation values ​​of the microsphere sample group 3 months after surgery were lower than those of the control group and the buffer group, and as the concentration of the microspheres increased, the trabecular separation gradually decreased (the differences between the groups were statistically significant).

[0066] Table 2 Comparison of intertrochanteric trabecular histomorphology of different samples 3 months after surgery (n=8)

[0067]

[0068] Example 5 Experimental study on the effect of poly(lactic-co-glycolic acid) PLGA microsphere solution on chondrocytes

[0069] 1. Chondrocyte Extraction and Culture

[0070] The chondrocytes are obtained from the cartilage tissue of the ears of three-week-old New Zealand white rabbits, and are cultured and passaged once every one week or so. The present invention uses the third generation chondrocytes for the planting of microsphere materials.

[0071] 2. MTT method to test the effect of PLGA microsphere solution on the proliferation of chondrocytes

[0072] PBS buffer was used as the blank control group, and PLGA microsphere solutions with mass concentrations of 10%, 20%, and 40% were prepared with PBS buffer as the experimental groups. First, the samples of the above groups were added to a 96-well plate, with 100 μL of microsphere solution in each well, and 5 parallel wells were set for each sample. Then, 100 μL of 1×10 4 / mL of cell suspension was placed in a cell culture incubator. After incubation in the dark for 2, 4, 6 and 8 days, 20 μL of MTT solution was added to each well. After incubation in the dark for 4 hours, the supernatant in each well was removed and then dissolved into a transparent solution with 150 μL of dimethyl sulfoxide. The reading at 570 nm was tested by an ELISA instrument, and the proliferation results of chondrocytes on microsphere samples of different concentrations were finally calculated. The polylactic acid-co-glycolic acid microspheres were prepared in Example 2.

[0073] Test results such as Figure 4 As shown, compared with the blank control group, different concentrations of PLGA microsphere solutions can promote the proliferation of chondrocytes; and, with the increase of culture time and microsphere concentration, the number of chondrocytes in the sample group also gradually increased, indicating that PLGA microspheres can promote the proliferation of chondrocytes.

[0074] Example 6 Effect of poly(lactic-co-glycolic acid) PLGA microsphere solution on the growth of cartilage tissue

[0075] 1. Isolation of Chondrocytes

[0076] Cut the pig auricle cartilage of about 2cm×2cm under sterile conditions. After removing the skin and subcutaneous tissue under sterile conditions, remove the perichondrium and cut into pieces of about 1mm 3 The cartilage blocks were digested with 0.25% trypsin for 40 minutes, and then 5 times the volume of 0.2% collagenase was added and digestion continued for 16-24 hours. After filtering the cells with a cell filter, the precipitate was collected by repeated centrifugation and finally placed in a constant temperature incubator for culture to obtain P0 generation pig ear chondrocytes.

[0077] 2. Mixing of Chondrocytes and PLGA Microspheres

[0078] PBS buffer was used to prepare PLGA microsphere solutions with a mass concentration of 10% and 40% as the experimental group. P2 generation pig ear chondrocytes were selected with a density of 1×10 8 / mL of cell suspension. Add appropriate amount of cell suspension, PLGA microsphere solution and culture medium into 6-well plate and continue to culture in constant temperature incubator. Specific grouping is shown in Table 3.

[0079] Table 3 Specific composition information of group A and group B

[0080]

[0081] 3. Real-time PCR

[0082] After the cell microspheres were cultured in vitro for 4 weeks, the expression of cartilage-related genes was detected by real-time PCR, and the expression level of each group relative to the internal reference gene GAPDH was statistically analyzed.

[0083] 1) Reaction system: 0.3 μl of upstream and downstream primers, 5 μl of SYBR, 0.4 μl of template, and 4 μl of ddH2O.

[0084] 2) Reaction procedure: 95°C for 1 min, 95°C for 5 s, 60°C for 1 min, and the number of cycles was 40.

[0085] 3) Quantitative analysis was performed using GAPDH in each group as an internal reference.

[0086] 4) The primer sequences are shown in Table 4.

[0087] Table 4 Real-time PCR primer sequences

[0088]

[0089] Figure 5 The results of the test of the relative expression of cartilage-related genes after in vitro culture of microsphere-chondrocyte complexes show that PLGA microspheres contribute to cartilage formation. Compared with low-concentration microsphere samples, the expression of cartilage-related genes in high-concentration microsphere samples was significantly increased, and the difference was statistically significant.

[0090] Example 7 Effect of poly(lactic-co-glycolic acid) PLGA microsphere solution on cartilage tissue growth

[0091] 1. In vivo injection of microsphere solution

[0092] Three healthy Bama miniature pigs were taken, male, 2 months old, weighing 8-10 kg. After anesthesia and disinfection, the cavity was separated from the pig's subcutaneous cavity according to the aseptic operation process. PBS buffer solution was used as the control group, and PLGA microsphere solutions with mass concentrations of 20% and 60% respectively prepared with PBS buffer solution were used as the sample group. 1 mL of the above control group and sample group were injected into the subcutaneous tissue on both sides of the pig's back, and sutured with sutures and marked. The material was taken out in the second week after injection and placed in formalin for histological staining.

[0093] 2. Histological Staining

[0094] (1) Tissue section preparation

[0095] 1) The tissue fixed with 10% formalin was trimmed into tissue blocks of appropriate size.

[0096] 2) Dehydration in gradient ethanol: 70% ethanol for 30 minutes, 80% ethanol for 30 minutes, 90% ethanol for 30 minutes, 95% ethanol I for 30 minutes, 95% ethanol II for 30 minutes, anhydrous ethanol I for 40 minutes, anhydrous ethanol II for 40 minutes, anhydrous ethanol: xylene (1:1) for 40 minutes, and xylene for 8 minutes twice.

[0097] 3) After taking out from xylene, quickly put into paraffin: xylene (1:1) for 40 minutes, soft wax with a melting point of 50-52°C for 40 minutes, and hard wax with a melting point of 60-62°C for 40 minutes.

[0098] 4) Embed the tissue in hard wax and leave it at room temperature overnight.

[0099] 5) The slice thickness of the tissue slicer is 5-8μm.

[0100] 6) 45℃ distilled water spread.

[0101] 7) Bake the slices at 50℃ for 1.5-2 hours and set aside.

[0102] 3. Immunohistochemical staining of type II collagen

[0103] 1) After the frozen sections were allowed to recover at room temperature for 1 hour, they were fixed with 4% paraformaldehyde for 15 minutes and then washed with PBS for 3 times, 3 minutes each time.

[0104] 2) Perform antigen retrieval according to the procedure of the pepsin antigen retrieval kit, add hydrogen peroxide methanol solution and incubate at room temperature for 10 minutes, and wash with PBS three times, each time for 3 minutes.

[0105] 3) Incubate with pepsin for 30 minutes and wash with PBS three times, 3 minutes each time.

[0106] 4) Follow the steps of the immunohistochemistry kit and block the endogenous enzymes with 3% hydrogen peroxide solution at room temperature.

[0107] 5) Wash with PBS three times, 3 minutes each time.

[0108] 6) Nonspecific antigens were blocked with 10% goat serum for 10 minutes.

[0109] 7) Dilute the primary antibody with PBS according to the instructions, discard the goat serum, add the primary antibody, and incubate at 4°C overnight.

[0110] 8) Equilibrate at room temperature for 1-2 hours.

[0111] 9) Discard the primary antibody and wash with PBS three times, 3 minutes each time.

[0112] 10) Add secondary antibody and incubate at 37°C for 30 minutes.

[0113] 11) Wash with PBS three times, 3 minutes each time.

[0114] 12) Prepare DAB colorimetric solution according to the instructions, add the solution under a microscope, and stop color development when a positive result appears.

[0115] 13) Stain with hematoxylin for 2 minutes and wash with water.

[0116] 14) Soak in tap water for 10 minutes.

[0117] 15) Dehydrate in anhydrous ethanol for 5 minutes.

[0118] 16) Transparent the slides with xylene I for 5 minutes and with xylene II for 5 minutes, and seal the slides with neutral gum.

[0119] Immunohistochemistry results Figure 6 The results showed that after 2 weeks of in vivo injection, compared with the PBS buffer solution group, the PLGA microsphere solution sample group had obvious positive expression of type II collagen, and the statistical results showed that the expression of type II collagen in the high-concentration microsphere solution was significantly higher than that in the low-concentration microsphere solution. The results show that the PLGA microsphere solution can promote cartilage growth.

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. Application of polylactic acid and its copolymers, polylactic acid microspheres, and polylactic acid copolymer microspheres in the preparation of drugs for promoting the growth of bone tissue and cartilage tissue, wherein: The molecular weight of the polylactic acid and its copolymer is 400Da-300kDa; Preferably, the molecular weight of the polylactic acid and its copolymer is 5000Da-100kDa; for example, 5000Da-20kDa, 15000Da.

2. The use according to claim 1, characterized in that The polylactic acid includes L-polylactic acid, D-polylactic acid and racemic polylactic acid, and is preferably L-polylactic acid.

3. The use according to claim 1, 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, polylactic acid-chitosan copolymer, and lactide-caprolactone copolymer; Preferably, the polylactic acid copolymer is a polylactic acid-glycolic acid copolymer.

4. The use according to any one of claims 1 to 3, characterized in that: The preparation method of polylactic acid microspheres and polylactic acid copolymer microspheres comprises the following steps: 1) mixing polylactic acid or a polylactic acid copolymer and a solvent to obtain a polylactic acid solution or a polylactic acid copolymer solution; Preferably, the solvent is any one of dichloromethane, chloroform, ethyl acetate, acetone or toluene, or a mixture of any two or more thereof; 2) Mixing the polylactic acid solution or the polylactic acid copolymer solution with the polyvinyl alcohol aqueous solution, emulsifying at high speed, stirring to remove the solvent, and freeze-drying to obtain polylactic acid microspheres.

5. The use according to claim 4, characterized in that In step 1), the mass volume ratio of polylactic acid or polylactic acid copolymer to solvent is 1g:5mL-1g:30mL, preferably 1g:10mL-1g:20mL, for example 1g:15mL; and / or, in step 2), the mass concentration of the polyvinyl alcohol aqueous solution is 0.05%-5.0%, preferably 0.1%-2.0%, for example 0.5%; And / or, in step 2), the volume ratio of the polylactic acid solution or the polylactic acid copolymer solution to the polyvinyl alcohol aqueous solution is 1:5-1:30, preferably 1:10-1:20, for example 1:13, 1:15; And / or, in step 2), the rotation speed of the high-speed emulsification is 1000 rpm / min-5000 rpm / min, for example 3000 rpm / min; And / or, in step 2), the emulsification time is 5-20 minutes, for example 10 minutes.

6. The use according to any one of claims 1 to 5, characterized in that: When administered, the mass concentration of polylactic acid and its copolymers, polylactic acid microspheres, and polylactic acid copolymer microspheres is 1%-80%, preferably 5%-50%, such as 10%, 20%, 30%, 40%.

7. The use according to any one of claims 1 to 6, characterized in that: The drug may optionally further include excipients; Preferably, the auxiliary material includes at least one of a stabilizer, a filler, a binder, a surfactant and a lubricant; Preferably, the surfactant is selected from one or more of polyethylene glycol, sodium dodecyl sulfate, Tween, and Span; Preferably, the stabilizer is selected from one or both of carboxymethyl cellulose 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 hydroxypropyl cellulose, methyl cellulose, sodium hyaluronate, collagen, and polyvinyl pyrrolidone; Preferably, the lubricant is selected from one or more of magnesium stearate, calcium stearate and stearic acid.

8. The use according to any one of claims 1 to 7, characterized in that: The drug may optionally be further used in combination with other active ingredients, which 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 collagen, gelatin, osteocalcin (Bone Gla Protein, BGP), bone morphogenetic protein (Bone morphogenetic protein, BMP), basic fibroblast growth factor (Basic Fibroblast Growth Factor, bFGF), insulin growth factor (Insulin Iike Growth Factor, IGF), transforming growth factor β (Transforming Growth Factor-β, TGF-β), platelet derived growth factor (Platelet derived growth factor, PDGF), concentrated growth factor fibrin (Concentrated Growth Facter, CGF) and platelet-rich fibrin (Platelet-Rich Fibrin, PRF), etc.

9. The use according to any one of claims 1 to 8, characterized in that: The administration methods of the drug include but are not limited to intravenous injection, in situ injection, intramuscular injection, subcutaneous injection, oral administration, application, etc.

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

Images