Use of polylactic acid-glycolic acid copolymer scaffold in promotion of chondrogenic differentiation and regeneration

Abstract

The use of a polylactic acid-glycolic acid copolymer scaffold in the promotion of chondrogenic differentiation and regeneration other than the diagnosis and treatment of diseases, wherein the polylactic acid-glycolic acid copolymer is PLGA (50:50). The polylactic acid-glycolic acid copolymer scaffold can promote the differentiation of chondrocyte precursors into cartilage and increase the formation of cartilage collagen matrix and chondroproteoglycan. A degradation and release liquid of the polylactic acid-glycolic acid copolymer scaffold can effectively promote the differentiation of chondrocyte precursors into cartilage and increase the formation of cartilage collagen matrix and chondroproteoglycan, thereby playing a role in promoting cartilage regeneration.

Description

Application of poly(lactic-co-glycolic acid) scaffold in promoting cartilage differentiation and regeneration Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a novel degradable and drug-loaded cartilage regeneration promoting material. Background Art

[0002] With the increasing aging of the population, degenerative diseases have become a major challenge for society and healthcare. Data show that approximately 50% of people over 65 face osteoarthritis (OA), and knee OA is the leading cause of chronic pain and disability among the elderly. As one of the diseases with the highest disability rates, knee OA places a heavy burden on the global economy. As a general joint disease, OA patients present with cartilage damage, synovial inflammation, and abnormal subchondral bone remodeling. The poor self-repair capacity of articular cartilage and the inflammatory environment pose significant challenges to cartilage regeneration. Studies have shown that during OA disease progression, the structural integrity of the cartilage-bone interface (CBI) is disrupted, allowing blood vessels from the subchondral bone to invade the cartilage area and mediators and factors within the joint cavity to penetrate into the bone tissue, subsequently causing damage to both the cartilage and subchondral bone.

[0003] Poly(lactic-co-glycolic acid) (PLGA) is a polymer formed by cross-linking poly(lactic acid) (PLA) and poly(glycolic acid) (PGA). It is a biodegradable implant material approved by the US Food and Drug Administration (FDA) and exhibits excellent cell compatibility and drug loading properties. The in vivo degradation rate of PLGA polymer is related to the cross-linking polymerization ratio of PLA and PGA. Studies have found that PLGA with a 50:50 PLA:PGA ratio exhibits strong hydrophilicity, low crystallinity, and low mechanical strength and elastic modulus.

[0004] Based on this, the applicant used a PLGA (50:50) polymer as a carrier and low-temperature 3D printing technology to prepare a three-dimensional porous PLGA (50:50) scaffold for promoting cartilage regeneration. The inventors discovered that PLGA (50:50) can not only effectively load various natural small molecule drugs, but also promotes cartilage matrix formation, participating in cartilage differentiation and regeneration. The release liquid from the PLGA (50:50) scaffold can transform into a cartilage microenvironment, thereby promoting cartilage regeneration. This novel application has not yet been reported in any papers, publications, or patents.

[0005] Summary of the Invention

[0006] In view of the defects existing in the above-mentioned prior art, the purpose of the present invention is to design and provide a polylactic acid-glycolic acid copolymer scaffold for use in promoting cartilage differentiation and regeneration. The present invention adopts low-temperature 3D printing technology to prepare a new material for promoting cartilage regeneration, namely PLGA (50:50) scaffold. The present invention adopts mouse cartilage precursor cell (ADTC5 cells) chondrogenesis induction experiment to evaluate the effect of the degradation liquid of the new degradable biomaterial on promoting cartilage regeneration. The results showed that the degradation release liquid of the PLGA (50:50) scaffold material can effectively promote the differentiation of mouse cartilage precursor cells into cartilage, and increase the formation of cartilage collagen matrix and cartilage proteoglycan. The PLGA (50:50) scaffold decomposition release liquid can change into the cartilage microenvironment, thereby playing a role in promoting cartilage regeneration.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In one aspect, the present invention provides a use of a poly(lactic-co-glycolic acid) scaffold for non-disease diagnosis and treatment purposes in promoting cartilage differentiation and regeneration.

[0009] In the above-mentioned use, the polylactic acid-glycolic acid copolymer is PLGA (50:50).

[0010] The polylactic acid-glycolic acid copolymer scaffold can promote the differentiation of cartilage precursor cells into cartilage and increase the formation of cartilage collagen matrix and cartilage proteoglycan.

[0011] In the above-mentioned use, the polylactic acid-co-glycolic acid scaffold can promote the formation of cartilage matrix and accelerate the differentiation of ATDC5 cells into chondrogenic cells.

[0012] The polylactic acid-glycolic acid copolymer scaffold can enhance cell activity.

[0013] In a second aspect, the present invention provides a method for using the aforementioned use, comprising contacting the PLGA (50:50) scaffold degradation release liquid with at least one cell having chondrogenic potential.

[0014] In the method of use, the PLGA (50:50) scaffold degradation release liquid is the first extract and / or the second extract.

[0015] The method for using the described purpose, the preparation process of the first extract is: using 1,4-dioxane as a solvent and PLGA (50:50) as a solute, preparing a PLGA (50:50) scaffold by low-temperature 3D printing technology; after irradiation sterilization, the PLGA (50:50) scaffold is soaked in serum-free MEM culture medium at a ratio of 0.1g / mL, and after soaking on a shaker at room temperature for 24 hours, the culture medium is collected to obtain the first extract; preferably, the first extract is obtained by collecting the culture medium and then diluting it 5-10 times.

[0016] The second extract is prepared by soaking the PLGA (50:50) scaffold from which the first extract was extracted in serum-free MEM medium at a ratio of 0.1 g / mL. After soaking on a shaker at room temperature for 24 hours, the medium is collected and diluted 5-10 times to obtain the second extract. Preferably, the second extract is obtained by diluting the collected medium 5-10 times.

[0017] In a third aspect, the present invention provides use of a poly(lactic-co-glycolic acid) scaffold in the preparation of a drug for promoting cartilage differentiation and regeneration.

[0018] In a fourth aspect, the present invention provides a pharmaceutical composition for promoting cartilage differentiation and regeneration, comprising a PLGA (50:50) scaffold and a small molecule drug loaded thereon.

[0019] The design concept of this invention is to use low-temperature 3D printing technology to transform disordered PLGA (50:50) raw materials into an ordered, porous, and well-connected three-dimensional scaffold. The following experiments detail the new application of the degradable PLGA (50:50) biomaterial:

[0020] (1) 3D printing "ink" was prepared using 1,4-dioxane as solvent and PLGA (50:50) as solute. PLGA (50:50) raw material was used to prepare tissue engineering scaffolds with suitable structural properties through low-temperature 3D printing technology. Micro-CT was then used to characterize the PLGA (50:50) scaffold. The results showed that the pore structure of the scaffold was uniform and the scaffold had good connectivity.

[0021] (2) After irradiation sterilization, the PLGA (50:50) scaffold was soaked in serum-free MEM medium at a ratio of 0.1 g / ml. After soaking on a shaker at room temperature for 24 hours, the medium was collected as the first extract. Then, the scaffold was soaked in serum-free MEM medium at a ratio of 0.1 g / ml. After soaking on a shaker at room temperature for another 24 hours, the medium was collected as the second extract.

[0022] This study used a chondrogenic induction experiment with mouse ADTC5 cartilage precursor cells to evaluate the biosafety and cartilage regeneration efficacy of the PLGA (50:50) scaffold degradation solutions (primary and secondary extracts). The results showed that the release of the degradation solution effectively promoted cartilage differentiation of mouse cartilage precursor cells and increased the formation of cartilage collagen matrix and cartilage proteoglycans. These results confirm the novel application of the biodegradable PLGA (50:50) scaffold described in this invention.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The release fluid from the degradation of the PLGA (50:50) scaffold material effectively promotes the differentiation of cartilage precursor cells into cartilage and increases the formation of cartilage collagen matrix and cartilage proteoglycans. The release fluid from the degradation of the PLGA (50:50) scaffold modifies the cartilage microenvironment, thereby promoting cartilage regeneration. The release fluid from the degradation of the PLGA (50:50) scaffold promotes cartilage matrix formation and accelerates the chondrogenic differentiation of ATDC5 cells. Furthermore, a chondrogenic differentiation induction solution containing the first and / or second extracts can enhance cell activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 shows the molecular formula of PLGA (50:50);

[0026] Figure 2 shows the structural characterization of the PLGA (50:50) scaffold;

[0027] Figure 3 shows the biosafety assessment of the release solution from the degradation of PLGA (50:50) scaffolds. (A) Biosafety of the first extract of the PLGA (50:50) scaffold; (B) Biosafety of the second extract of the PLGA (50:50) scaffold; (C) Biosafety of the first and second extracts of the PLGA (50:50) scaffold in a chondrogenic differentiation system. Experimental data are expressed as mean ± SD, n = 6. Graphpad Prism 6 software (Graphpad Software Inc, San Diego, CA, USA) was used for data processing and statistical analysis was performed using ANOVA test. P < 0.05 was considered statistically significant.

[0028] FIG4 is a graph showing the effect of PLGA (50:50) scaffold degradation release fluid on promoting ATDC5 cell differentiation into chondrogenic cartilage.

[0029] DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0031] The poly(lactic-co-glycolic acid) copolymer (50:50) in this invention is chemically synthesized with poly(lactic acid) (PLA) and poly(glycolic acid) (PGA) as its basic molecular backbone in a 50:50 ratio. Its English name is Poly (lactic-co-glycolic acid) (50:50), abbreviated as PLGA(50:50), and its chemical formula is (C3H6O3.C2H4O3)n, as shown in Figure 1. The PLGA(50:50) used in this invention was purchased from Shandong Academy of Pharmaceutical Sciences with CAS number 34346-01-5, batch number 21071601, molecular weight 96,000, and viscosity 0.71 dL / g.

[0032] Example 1: PLGA (50:50) scaffold preparation and characterization

[0033] Weigh 10 g of PLGA (50:50) and place it in a 100 mL beaker. Add 40 mL of 1,4-dioxane and stir for 12 hours. The stirring method is to stir mechanically at room temperature until the solute is fully dissolved and dispersed. Prepare 3D printing "ink" with PLGA (50:50) as the solute. Stir and mix thoroughly before 3D printing. After the temperature of the 3D printer is pre-cooled to -30°C, the printing parameters are set as follows: size 40 mm × 40 mm × 5 mm, line spacing 0.45 mm, layer height 0.15 mm, nozzle movement speed 20.0 mm / s; material filling 1.0 mm 3 / s, and the inner diameter of the printhead was 400 μm. After printing, the product was placed in a freeze dryer and freeze-dried under vacuum for 72 hours to remove the organic solvent. The PLGA (50:50) scaffold was scanned using a Skyscan 1176 micro-CT with the following scanning parameters: voltage: 40 kV, current: 497 μA, resolution: 9 μm, no filter. The front and side pore sizes were analyzed after scanning. The results are shown in Figure 2: front pore diameter: 175.7 ± 7.7 μm; side pore diameter: 191.3 ± 8.8 μm; and porosity: 37.2 ± 5.6%.

[0034] Example 2: Biocompatibility test of PLGA (50:50) scaffold degradation solution on ATDC5 cells in vitro

[0035] Preparation of the scaffold degradation release solution: PLGA (50:50) scaffolds were sterilized by irradiation at 15 kGy and extracted in vitro at a concentration of 0.1 g / ml (scaffold weight / extraction solution volume) in MEM medium containing 10% serum and 1% double-antibody antibodies. After soaking in the extraction solution, the scaffolds were sealed and shaken on a shaker at room temperature for 24 hours, using sterile procedures throughout. After 24 hours, the extract was collected and designated the first extract. The same volume of extraction MEM medium was then added and the above process repeated. After 24 hours, the extract was collected and designated the second extract.

[0036] All extracts were filtered with a 0.22 μm filter head, and the filtrate was placed in a refrigerator at 4 degrees for later use. The filtered extract was diluted with MEM culture medium containing 10% serum and 1% double antibody to obtain extracts with dilution ratios of 0 times, 5 times, 10 times, and 20 times. Then, mouse chondrogenic cell line (ATDC5 cells) was used for cell safety analysis. Specific operation: ATDC5 cells were cultured with DMEM / F12 culture medium containing 10% serum and 1% double antibody. Cells were seeded in 96-well plates at a density of 0.5 million cells per well. After culturing for 2 days, the growth medium containing the extract was replaced and the cells were cultured for 48 hours. The experiment was divided into normal group (Normal), 0 times (0), 5 times (5), 10 times (10), and 20 times (20). At the endpoint, the extraction medium was removed, and 10% CCK-8 reagent was added to each well for a further 3 hours of incubation. OD values ​​were measured at 490 nm and normalized to those of the Normal group. As shown in Figure 3, both the original solutions of the first and second extractions (i.e., the 0-fold dilution group) exhibited significant cytotoxicity.

[0037] Therefore, the present invention screened the 5-fold (5) and 10-fold (10) dilutions for subsequent chondrogenic differentiation experiments. Specifically, after the extract was diluted 5-fold and 10-fold, 10 ng / mL TGF-β, 10% ITS premix, 0.1 μM dexamethasone (Dex), 1% sodium pyruvate, and 1 μM vitamin C phosphate were added to prepare chondrogenic differentiation induction fluid. ATDC5 cells were seeded in a 96-well plate at a density of 0.5 million cells per well. After culturing for 2 days, the diluted chondrogenic differentiation induction fluid containing the extract was replaced and the cells were cultured for 48 hours. The experiment was divided into a chondrogenic differentiation control group (Control), a 5-fold (5) dilution, and a 10-fold (10) dilution. At the end point, the extract culture fluid was removed, and 10% CCK-8 reagent was added to each well and incubated for another 3 hours. The OD value was detected at 490 nm, and the OD value of each group was normalized to the OD value of the Normal group. The results showed that in the chondrogenic differentiation system, the chondrogenic differentiation induction medium containing the extract could enhance cell activity.

[0038] Example 3: Effect of PLGA (50:50) scaffold degradation solution on chondrogenic differentiation of ATDC5 cells in vitro

[0039] Based on the results of Example 2, the present invention used 5-fold and 10-fold diluted extracts for chondrogenic differentiation experiments. Specifically, after the extract was diluted 5-fold and 10-fold, 10 ng / mL TGF-β, 10% ITS premix, 0.1 μM dexamethasone (Dex), 1% sodium pyruvate, and 1 μM vitamin C phosphate were added to prepare chondrogenic differentiation induction solutions, respectively. ATDC5 cells were seeded at a concentration of 100,000 cells per well in a 12-well plate. After two days of culture, the chondrogenic differentiation induction solution containing the diluted extract was replaced with the diluted extract and the cells were cultured for another eight days, with the chondrogenic differentiation induction solution replaced every two days. The experiment was divided into a normal growth culture group (Normal), a chondrogenic differentiation control group (Control), and a PLGA (50:50) scaffold extract diluted 5x (5×) and 10x (10×) groups. At the end of the experiment, cells were fixed with 4% paraformaldehyde (neutral) for 30 minutes, then washed three times with sterile double-distilled water and stained with 1% Alcian Blue working solution at room temperature in the dark overnight. The next day, the stain was discarded, cells were washed three times with sterile double-distilled water, and photographed under a white light microscope. As shown in Figure 4, the control group, the first extract, and the second extract diluted 5x (PLGA 5×) and 10x (PLGA 10×) all promoted cartilage matrix formation and accelerated chondrogenic differentiation of ATDC5 cells. The second extract had a more potent effect, resulting in greater cartilage matrix formation.

Claims

1. Use of a polylactic acid-glycolic acid copolymer scaffold for non-disease diagnosis and treatment purposes in promoting cartilage differentiation and regeneration.

2. The use according to claim 1, characterized in that The polylactic acid-glycolic acid copolymer is PLGA (50:50).

3. The use according to claim 1, characterized in that The polylactic acid-glycolic acid copolymer scaffold can promote the differentiation of cartilage precursor cells into cartilage and increase the formation of cartilage collagen matrix and cartilage proteoglycan.

4. The use according to claim 1, characterized in that The polylactic acid-glycolic acid copolymer scaffold can promote the formation of cartilage matrix and accelerate the differentiation of ATDC5 cells into chondrogenesis.

5. The use according to claim 1, characterized in that The poly(lactic-co-glycolic acid) scaffold can enhance cell activity.

6. The method of use according to claim 2, characterized in that: The PLGA (50:50) scaffold degradation release liquid is contacted with at least one cell with chondrogenic potential.

7. The method of use according to claim 6, characterized in that: The PLGA (50:50) scaffold degradation release liquid is the first extract and / or the second extract.

8. The method of use according to claim 7, characterized in that: The preparation process of the first extract is as follows: using 1,4-dioxane as a solvent and PLGA (50:50) as a solute, preparing a PLGA (50:50) scaffold by low-temperature 3D printing technology; after the PLGA (50:50) scaffold is irradiated and sterilized, it is soaked in a serum-free MEM culture medium at a ratio of 0.1 g / mL, and after soaking on a shaker at room temperature for 24 hours, the culture medium is collected to obtain the first extract; The preparation process of the second extract is as follows: the PLGA (50:50) scaffold after extracting the first extract is then soaked in serum-free MEM culture medium at a ratio of 0.1 g / mL, and after soaking on a shaker at room temperature for 24 hours, the culture medium is collected to obtain the second extract.

9. Use of a polylactic acid-glycolic acid copolymer scaffold in the preparation of drugs for promoting cartilage differentiation and regeneration.

10. A pharmaceutical composition for promoting cartilage differentiation and regeneration, characterized in that: It comprises a PLGA (50:50) scaffold and a small molecule drug loaded thereon.

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