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

Abstract

The use of a polylactic-co-glycolic acid scaffold in promoting cartilage differentiation and regeneration belongs to the technical field of biomedical materials. The present invention includes the use of a polylactic-co-glycolic acid scaffold for non-disease diagnosis and treatment purposes in promoting cartilage differentiation and regeneration. The polylactic-co-glycolic acid copolymer is PLGA (50:50). The polylactic-co-glycolic acid scaffold can promote the differentiation of cartilage precursor cells into cartilage, and increase the formation of cartilage collagen matrix and cartilage proteoglycans. The degradation release liquid of the PLGA (50:50) scaffold material can effectively promote the differentiation of cartilage precursor cells into cartilage, and increase the formation of cartilage collagen matrix and cartilage proteoglycans. The release liquid of the PLGA (50:50) scaffold can be changed into a cartilage microenvironment, thereby playing a role in promoting cartilage regeneration.

Description

Technical Field

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

[0002] As the trend of population aging intensifies, degenerative diseases have become a major challenge facing society and medicine. Data show that about 50% of the population over 65 years old is troubled by osteoarthritis (OA), and knee OA is the main cause of chronic pain and disability in the elderly. As one of the diseases with the highest disability rate, knee OA poses a heavy burden on the global economy. As an overall joint disease, OA patients show cartilage damage, synovial inflammation and abnormal subchondral bone reconstruction. The poor self-repair ability of articular cartilage and the inflammatory environment pose a huge challenge to cartilage regeneration. Studies have shown that in the course of OA disease, the structural integrity of the cartilage-bone interface (CBI) is destroyed, causing blood vessels in the subchondral bone to invade the cartilage area, and the media and factors in the joint cavity also penetrate into the bone tissue, which then causes damage to both the cartilage and the subchondral bone.

[0003] Polylactic acid-glycolic acid copolymer (PLGA) is a high molecular polymer formed by cross-linking polylactic acid (PLA) and polyglycolic acid (PGA). It is a degradable implant material approved by the US Food and Drug Administration (FDA) and has good cell compatibility and drug loading performance. The degradation rate of PLGA polymer in vivo is related to the cross-linking polymerization ratio of PLA and PGA. Studies have found that PLGA with a mass ratio of PLA and PGA of 50:50 has strong hydrophilicity, low crystallinity, low mechanical strength and elastic modulus.

[0004] Based on this, the applicant used PLGA (50:50) polymer material as a carrier and adopted low-temperature 3D printing technology to prepare a three-dimensional porous PLGA (50:50) scaffold that can be used to promote cartilage regeneration. The present invention found that PLGA (50:50) can not only effectively load various natural small molecule drugs, but also promote the formation of cartilage matrix and participate in cartilage differentiation and regeneration. The release liquid of PLGA (50:50) scaffold can be changed into a cartilage microenvironment, thereby playing a role in promoting cartilage regeneration. There are currently no reports on this new use in papers, books, and patents. Summary of the invention

[0005] In view of the defects existing in the above-mentioned prior art, the purpose of the present invention is to design and provide the use of polylactic acid-glycolic acid copolymer scaffold 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 show that the degradation release liquid of 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 release liquid of PLGA (50:50) scaffold can be changed into a cartilage microenvironment, thereby playing a role in promoting cartilage regeneration.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In one aspect, the present invention provides a use of a poly(lactic-co-glycolic acid) copolymer scaffold for non-disease diagnosis and treatment purposes in promoting cartilage differentiation and regeneration.

[0008] In the above-mentioned use, the polylactic acid-glycolic acid copolymer is PLGA (50:50).

[0009] 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.

[0010] In the use, the polylactic acid-glycolic acid copolymer scaffold can promote the formation of cartilage matrix and accelerate the differentiation of ATDC5 cells into chondrogenesis.

[0011] The use described above, the polylactic acid-glycolic acid copolymer scaffold can enhance cell activity.

[0012] In a second aspect, the present invention provides a method for using the aforementioned use, wherein the PLGA (50:50) scaffold degradation release liquid is contacted with at least one cell having chondrogenic potential.

[0013] In the method of using the application, the PLGA (50:50) scaffold degradation release liquid is the first extract and / or the second extract.

[0014] The method for using the 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 the PLGA (50:50) scaffold is irradiated and sterilized, it is soaked in a 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.

[0015] 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 medium at a ratio of 0.1 g / mL, and 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 collecting the medium and then diluting it 5-10 times.

[0016] In a third aspect, the present invention provides use of a poly(lactic acid-co-glycolic acid) copolymer scaffold in the preparation of a drug for promoting cartilage differentiation and regeneration.

[0017] 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.

[0018] The design idea of ​​the present invention is: the present invention uses low-temperature 3D printing technology to prepare disordered PLGA (50:50) raw materials into an ordered, porous, well-connected three-dimensional scaffold. The following experiment describes in detail the new use of degradable PLGA (50:50) biomaterials:

[0019] (1) Using 1,4-dioxane as solvent and PLGA (50:50) as solute, 3D printing "ink" was used to prepare PLGA (50:50) raw material into a tissue engineering scaffold 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.

[0020] (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 again. After soaking on a shaker at room temperature for another 24 hours, the medium was collected as the second extract.

[0021] The present invention uses a mouse cartilage precursor cell ADTC5 chondrogenesis induction experiment to evaluate the biosafety and cartilage regeneration effect of the PLGA (50:50) scaffold degradation solution (first extract and second extract). The results show that the material degradation release solution can effectively promote the differentiation of mouse cartilage precursor cells into cartilage and increase the formation of cartilage collagen matrix and cartilage proteoglycan. Based on the above results, it is confirmed that the new use of the degradable biomaterial PLGA (50:50) scaffold described in the present invention is established.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The release liquid of the PLGA (50:50) scaffold material degradation can effectively promote the differentiation of cartilage precursor cells into cartilage and increase the formation of cartilage collagen matrix and cartilage proteoglycan. The release liquid of the PLGA (50:50) scaffold can change the microenvironment of cartilage, thereby playing a role in promoting cartilage regeneration. The release liquid of the PLGA (50:50) scaffold degradation can promote the formation of cartilage matrix and accelerate the differentiation of ATDC5 cells into chondrogenesis. In addition, the chondrogenic differentiation induction solution containing the first extract and / or the second extract can enhance cell activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the molecular formula of PLGA (50:50);

[0025] Figure 2 To characterize the structure of PLGA (50:50) scaffold;

[0026] Figure 3 The biosafety of the degradation release solution of PLGA (50:50) scaffolds was evaluated; (A) the biosafety of the first extract of PLGA (50:50) scaffolds; (B) the biosafety of the second extract of PLGA (50:50) scaffolds; (C) the biosafety of the first / second extracts of PLGA (50:50) scaffolds in the chondrogenic differentiation system; the experimental data were expressed as Mean±SD, n=6, Graphpad prism 6software (Graphpad Software Inc, San Diego, CA, USA) was used for data processing, and ANOVA test was used for statistical analysis, p<0.05 was considered statistically significant;

[0027] Figure 4 This is a graph showing the effect of PLGA (50:50) scaffold degradation release fluid on promoting ATDC5 cell differentiation into chondrogenesis. DETAILED DESCRIPTION

[0028] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0029] The polylactic acid-glycolic acid copolymer (50:50) in the present invention is obtained by chemical synthesis with polylactic acid (PLA) and polyglycolic acid (PGA) as the basic molecular skeleton and the component ratio is 50:50. English name: Poly (lactic-co-glycolic acid) (50:50), referred to as PLGA (50:50), chemical formula is (C3H6O3.C2H4O3)n, molecular formula is as follows Figure 1 The PLGA (50:50) used in this invention was purchased from Shandong Academy of Pharmaceutical Sciences, with CAS No.: 34346-01-5, Batch No.: 21071601, Molecular Weight: 96000, and Viscosity: 0.71 dL / g.

[0030] Example 1: PLGA (50:50) scaffold preparation and characterization

[0031] Weigh 10g PLGA (50:50) in a 100mL beaker, add 40mL 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 precooled to -30℃, the printing parameters are set as follows: size 40mm×40mm×5mm, line spacing 0.45mm, layer height 0.15mm, nozzle movement speed 20.0mm / s; material filling 1.0mm 3 / s, the inner diameter of the print head is 400μm. After printing, the product is placed in a freeze dryer and freeze-dried under vacuum conditions for 72 hours to remove the organic solvent in the material. The PLGA (50:50) scaffold was scanned using Skyscan1176micro-CT, with the following scanning parameters: voltage: 40kv, current: 497μA, resolution: 9μm, No filter; after scanning, the front and side pore sizes were analyzed. The results are as follows Figure 2 As shown, the front pore diameter is 175.7±7.7μm; the side pores are 191.3±8.8μm; and the porosity is 37.2±5.6%.

[0032] Example 2: Biocompatibility test of PLGA (50:50) scaffold in vitro degradation solution on ATDC5 cells

[0033] Preparation of the stent degradation release solution: The PLGA (50:50) stent was sterilized by 15KGy irradiation and extracted in vitro at 0.1g / ml (stent weight / extract volume). The extract was a MEM medium containing 10% serum and 1% double-antibody. After the stent was soaked in the extract, it was sealed and shaken on a shaker at room temperature for 24 hours. The whole process was aseptic. After 24 hours, the extract was collected and named the first extract. Then continue to add the same volume of extraction MEM medium, repeat the above operation, and collect the extract after 24 hours, named the second extract.

[0034] All extracts were filtered with a 0.22 μm filter head, and the filtrate was placed in a 4-degree refrigerator for later use. The filtered extract was diluted with MEM culture medium containing 10% serum and 1% double antibody to obtain extracts with dilution multiples of 0, 5, 10, and 20. 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. The cells were inoculated in a 96-well plate at 5,000 cells per well. After 2 days of culture, 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 end point, the culture medium was removed, 10% CCK-8 reagent was added to each well and incubated for 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. Figure 3 As shown, the original solutions of the first and second extractions (ie, the 0-fold dilution group) both had significant cytotoxicity.

[0035] Therefore, the present invention screened the 5-fold (5) and 10-fold (10) releases for subsequent chondrogenic differentiation tests. The specific operation is that after the extract is diluted 5 times and 10 times, 10ng / mL TGF-β, 10% ITS premix, 0.1μM dexamethasone (Dex), 1% sodium pyruvate, and 1μM vitamin C phosphate are added to prepare chondrogenic differentiation induction fluid. ATDC5 cells are inoculated in a 96-well plate at 0.5 million cells per well. After culturing for 2 days, the diluted chondrogenic differentiation induction fluid containing the extract is replaced and the cells are cultured for 48 hours. The experiment is divided into a chondrogenic differentiation control group (Control), a 5-fold (5) release, and a 10-fold (10) release. At the end point, the extract culture fluid is removed, 10% CCK-8 reagent is added to each well and incubated for another 3 hours, and the OD value is detected at 490nm. The OD value of each group is normalized with 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.

[0036] Example 3: Effect of PLGA (50:50) scaffold degradation solution on chondrogenic differentiation of ATDC5 cells in vitro

[0037] Based on the results of Example 2, the present invention uses 5-fold and 10-fold extracts for chondrogenic differentiation test. The specific operation is: after the extract is diluted 5 times and 10 times, 10ng / mL TGF-β, 10% ITS premix, 0.1μM dexamethasone (Dex), 1% sodium pyruvate, and 1μM vitamin C phosphate are added to prepare chondrogenic differentiation induction solution. ATDC5 cells are inoculated in a 12-well plate at 100,000 cells per well. After culturing for 2 days, the chondrogenic differentiation induction solution containing the diluted extract is replaced and the cells are continued to be cultured for 8 days, and the chondrogenic differentiation induction solution is replaced every 2 days. The experiment is divided into a normal growth culture group (Normal), a chondrogenic differentiation control group (Control), and a PLGA (50:50) scaffold extract diluted 5 times (5×) and 10 times (10×) group. At the end of the experiment, the 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 overnight in the dark. The next day, the stain was discarded, the cells were washed three times with sterile double distilled water, and photographed under a white light microscope. Figure 4 As shown in the figure, the control group, the first extraction and the second extraction solution diluted 5 times (PLGA 5×) and 10 times (PLGA 10×) can promote the formation of cartilage matrix and accelerate the differentiation of ATDC5 cells into chondrocytes. Among them, the second extraction solution has a better effect on promoting cartilage differentiation and has more cartilage matrix formation.

Claims

1. A use of a polylactic-co-glycolic acid copolymer scaffold for non-disease diagnosis and treatment purposes alone in promoting cartilage differentiation and regeneration; The polylactic acid-glycolic acid copolymer is PLGA (50:50), and the PLGA (50:50) scaffold is prepared by low-temperature 3D printing technology; 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.

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

3. The method of use according to claim 1, characterized in that: contacting the PLGA (50:50) scaffold degradation release liquid with at least one cell having chondrogenic potential; The PLGA (50:50) scaffold degradation release liquid is the first extract and / or the second extract; 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.

4. Use of a polylactic acid-co-glycolic acid copolymer scaffold alone in the preparation of a drug for promoting cartilage differentiation and regeneration; The polylactic acid-glycolic acid copolymer is PLGA (50:50), and the PLGA (50:50) scaffold is prepared by low-temperature 3D printing technology.

5. A pharmaceutical composition for promoting cartilage differentiation and regeneration, characterized in that: It is composed of a PLGA (50:50) scaffold and small molecule drugs loaded thereon; The small molecule drug is not used to promote cartilage differentiation and regeneration.

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