An anti-inflammatory chondrocyte regeneration promoting material, a preparation method and application thereof

Abstract

The application relates to an anti-inflammatory and cartilage regeneration promoting material, a preparation method and application thereof, and belongs to the technical field of biomedical materials. The anti-inflammatory and cartilage regeneration promoting material comprises a PLGA (50:50) polymer material, and a huperzine A derivative dimer loaded on the PLGA (50:50) polymer material through 3D printing. The application also provides the use of the anti-inflammatory and cartilage regeneration promoting material in the preparation of anti-inflammatory, cartilage protection or cartilage regeneration promoting drugs. The PLGA / A10E scaffold has good biocompatibility, the PLGA / A10E scaffold degradation release liquid can effectively promote the differentiation of cartilage precursor cells into cartilage, and increase the formation of cartilage collagen matrix and cartilage proteoglycan. The in-vitro degradation liquid of the PLGA / A10E scaffold can promote the expression of SOX9 under non-inflammatory conditions; under LPS-mediated inflammatory conditions, the expression of MMP-13 can be inhibited, thereby promoting the synthesis of cartilage matrix and inhibiting the degradation of cartilage matrix.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to an anti-inflammatory cartilage regeneration material, its preparation method, and its application. Background Technology

[0002] As a systemic joint disease, osteoarthritis (OA) manifests as cartilage damage, synovial inflammation, and abnormal subchondral bone remodeling. Poor cartilage self-repair capabilities and an inflammatory environment pose significant challenges to cartilage regeneration. Studies have demonstrated that during the progression of OA, the integrity of the cartilage-bone interface (CBI) is disrupted, allowing blood vessels from the subchondral bone to infiltrate the cartilage region. Mediators and factors within the joint cavity also infiltrate the bone tissue, subsequently damaging both cartilage and subchondral bone. Therefore, suppressing inflammation, repairing damaged cartilage, and maintaining the integrity of the subchondral bone structure are crucial for OA treatment. Catecholamines and cholinergic fibers innervate the synovium, trabecular bone, and periosteum. Research has found that bone and joint tissue cells can express receptors for sympathetic and parasympathetic neurotransmitters (norepinephrine / epinephrine and acetylcholine, respectively), thus responding to autonomic nerve stimulation. These cells can also synthesize neurotransmitters, causing local effects. Further research has revealed that stimulating the sympathetic nervous system can lead to bone loss and disrupt joint homeostasis. The cholinergic pathway acts as a "brake" on the innate immune system. Acetylcholinesterase inhibitors (AChEIs) can activate cholinergic anti-inflammatory activity, and by ingesting beta-blockers to inhibit the sympathetic nervous system, pain in OA patients can be reduced. Furthermore, AChEIs can control subchondral bone remodeling, making them a promising candidate for potent treatment of OA.

[0003] Huperzine A is a semi-terpenoid alkaloid derived from the fern *Huperzine aegyptiaca*. It is a highly effective, selective, and reversible inhibitor of acetylcholinesterase (AChE).

[0004] Polylactic acid-glycolic acid copolymer (PLGA) is a high-molecular-weight polymer formed by cross-linking polylactic acid (PLA) and polyglycolic acid (PGA). It is a biodegradable implantable material approved by the U.S. Food and Drug Administration (FDA) and exhibits good cell compatibility and drug loading capacity. The degradation rate of PLGA in vivo is related to the cross-linking polymerization ratio of PLA and PGA. Studies have found that PLGA with a PLA:PGA ratio of 50:50 exhibits strong hydrophilicity, low crystallinity, and low mechanical strength and elastic modulus.

[0005] The inventors of this application synthesized a novel hupyridone (10)-tacrine, namely A10E, using Huperzine A and tacaline (a commercially available drug that is a commonly used cholinesterase inhibitor) as the basic molecular framework. It was demonstrated that A10E can activate anti-inflammatory pathways, inhibit bone resorption, and promote bone formation, exhibiting multiple effects of anti-inflammatory, bone-strengthening, and joint-protecting properties. However, no reports have been found regarding A10E's ability to promote cartilage regeneration in an inflammatory environment. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention aims to design and provide an anti-inflammatory and cartilage regeneration-promoting material, its preparation method, and its application. This invention uses PLGA (50:50) polymer material as a carrier and employs low-temperature 3D printing technology to load huperzine A derivative dimer (A10E) onto PLGA (50:50), thus preparing a novel anti-inflammatory and cartilage regeneration-promoting material, namely a PLGA / A10E scaffold. This scaffold material releases PLGA degradation liquid and A10E as PLGA degrades, thereby exerting anti-inflammatory, cartilage-protective, and cartilage regeneration-promoting effects.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] An anti-inflammatory and cartilage regeneration material, wherein the anti-inflammatory and cartilage regeneration material is a PLGA / huperzine A derivative dimer scaffold, the PLGA / huperzine A derivative dimer scaffold comprising PLGA (50:50) polymeric material and huperzine A derivative dimers loaded thereon by 3D printing.

[0009] The preparation method of the anti-inflammatory and cartilage regeneration material includes the following steps:

[0010] (1) Weigh out the huperzine A derivative dimer and add dimethyl sulfoxide to dissolve it;

[0011] (2) Weigh PLGA (50:50), add 1,4-dioxane, stir, add the solution obtained in step (1), and mix well;

[0012] (3) 3D printing is performed. After completion, the product is obtained and placed in a freeze dryer for vacuum drying to obtain an anti-inflammatory and cartilage regeneration material, namely PLGA / huperzine A derivative dimer scaffold (i.e. PLGA / A10E scaffold).

[0013] In the preparation method described above, the mass-to-volume ratio of the huperzine A derivative dimer to dimethyl sulfoxide in step (1) is 30 mg: 6 μL.

[0014] In the preparation method described above, the mass-to-volume ratio of PLGA (50:50) to 1,4-dioxane in step (2) is 10g:40mL; the stirring time is 12 hours; and the stirring method is: mechanical stirring in a closed system at room temperature until the solute is fully dissolved and dispersed.

[0015] The preparation method described herein, in step (3), the 3D printing conditions are as follows: after pre-cooling the 3D printer to -30℃, the printing parameters are set as follows: size 40mm×40mm×5mm, line spacing 0.45mm, layer height 0.15mm, nozzle moving speed 20.0mm / s; material filling 1.0mm. 3 / s, printhead inner diameter 400μm.

[0016] In the preparation method described above, the vacuum drying time in step (3) is 72 hours.

[0017] The use of the aforementioned anti-inflammatory and cartilage regeneration-promoting material in the preparation of anti-inflammatory, cartilage-protecting, or cartilage-regenerating drugs.

[0018] As described above, the anti-inflammatory and cartilage regeneration-promoting material can promote the differentiation of cartilage precursor cells into cartilage and increase the formation of cartilage collagen matrix and cartilage proteoglycans.

[0019] The aforementioned use is that the anti-inflammatory cartilage regeneration material promotes the expression of SOX9 under non-inflammatory conditions.

[0020] As described above, the anti-inflammatory and cartilage regeneration-promoting material can inhibit the expression of MMP-13 under LPS-mediated inflammatory conditions, thereby promoting cartilage matrix synthesis and inhibiting cartilage matrix degradation.

[0021] This invention provides a detailed description of novel applications of biodegradable biomaterials loaded with huperzine M-dimer through the following experiments:

[0022] (1) Using 1,4-dioxane as solvent and PLGA(50:50) as solute as 3D printing "ink", tissue engineering scaffolds loaded with A10E were prepared by loading huperzine methyl dimer A10E into PLGA(50:50) material through low-temperature 3D printing technology. The PLGA / A10E scaffold was characterized by Micro-CT. The results showed that the scaffold had a uniform pore structure and good connectivity.

[0023] (2) After irradiation sterilization, the PLGA / A10E scaffold was soaked in serum-free MEM medium at a ratio of 0.1 g / mL on a shaker at room temperature for 24 hours. The medium was then collected as the first extract. The scaffold was then soaked again in serum-free MEM medium at a ratio of 0.1 g / mL on a shaker at room temperature for another 24 hours. The medium was then collected as the second extract. The A10E content in the first extract was calculated to be approximately 24 μM, and the A10E content in the second extract was approximately 21 μM.

[0024] (3) This invention uses a mouse chondrogenic induction experiment with ADTC5 precursor cells to evaluate the anti-inflammatory, cartilage-protective, and cartilage regeneration-promoting effects of the degradation solution (first and second extracts) of this novel biodegradable biomaterial. The results show that the degradation solution effectively promotes the differentiation of mouse chondrogenic precursor cells into chondrocytes and increases the formation of cartilage collagen matrix and proteoglycans. Furthermore, it was found that SOX9 expression is promoted under non-inflammatory conditions, while MMP-13 expression is inhibited under LPS-mediated inflammatory conditions. Based on these results, the novel use of the biodegradable biomaterial loaded with huperzine methyldimer A10E described in this invention is confirmed. Based on these results, the applicant confirms that the biodegradable biomaterial loaded with huperzine methyldimer of this invention has novel uses for anti-inflammatory, cartilage-protective, and cartilage regeneration-promoting effects.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The degradation and release fluid of the PLGA / A10E scaffold of the present invention has the effect of promoting cartilage regeneration in both non-inflammatory and inflammatory environments.

[0027] 2. The PLGA / A10E scaffold of this invention exhibits good biocompatibility, with an A10E loading efficiency of over 34.4%. Huperzine methyl dimer A10E is gradually released as PLGA degrades, with a 24-hour degradation release rate of approximately 12% or more.

[0028] 3. This invention uses a mouse chondrogenic ADTC5 cell induction experiment to evaluate the anti-inflammatory, cartilage-protective, and cartilage regeneration-promoting effects of this anti-inflammatory and cartilage-regenerating material. The results show that the PLGA / A10E scaffold degradation and release solution can effectively promote the differentiation of mouse chondrogenic cells into cartilage and increase the formation of cartilage collagen matrix and cartilage proteoglycans.

[0029] 4. The in vitro degradation solution of the PLGA / A10E scaffold can promote the expression of SOX9 under non-inflammatory conditions; under LPS-mediated inflammatory conditions, it can inhibit the expression of MMP-13, thereby promoting cartilage matrix synthesis and inhibiting cartilage matrix degradation. Attached Figure Description

[0030] Figure 1 The molecular formulas of huperzine methyl dimer A10E and PLGA (50:50) are:

[0031] Figure 2 A schematic diagram of the specific chemical synthesis route for huperzine methyl dimer A10E;

[0032] Figure 3 It is the huperzine methyl dimer A10E 1 H-NMR test results;

[0033] Figure 4 The chromatogram shows the huperzine methyl dimer A10E.

[0034] Figure 5 The activity of AChE inhibitors in huperzine methyl dimer A10E;

[0035] Figure 6 Here is a structural characterization diagram of the PLGA / A10E stent;

[0036] Figure 7 The peak area and standard curve for each concentration of A10E are shown.

[0037] Figure 8 The measured value of A10E in the PLGA / A10E support;

[0038] Figure 9 The peak area and standard curve of A10E at various concentrations under in vitro degradation conditions;

[0039] Figure 10 The detected value of A10E in the extract of the PLGA / A10E stent;

[0040] Figure 11 The study aimed to assess the biosafety of the degradation and release solutions of the PLGA / A10E scaffold; specifically, (A) the biosafety of the first extract of the PLGA / A10E scaffold; (B) the biosafety of the second extract of the PLGA / A10E scaffold; and (C) the biosafety of the first and second extracts of the PLGA / A10E scaffold in a chondrogenic differentiation system. Experimental data are expressed as Mean ± SD, n = 6. Data processing was performed using Graphpadprism 6 software (Graphpad Software Inc, San Diego, CA, USA), and statistical analysis was conducted using the ANOVA test. A p < 0.05 was considered statistically significant.

[0041] Figure 12 The effect of PLGA / A10E scaffold degradation release solution on promoting ATDC5 cell differentiation into chondrocytes under non-inflammatory conditions (n=2);

[0042] Figure 13 The effect of PLGA / A10E scaffold degradation release solution on promoting ATDC5 cell differentiation into chondrocytes under inflammatory conditions (n=2);

[0043] Figure 14 Effects of PLGA / A10E scaffold degradation release solution on SOX9 and MMP-13 proteins; (A) normal induction conditions without LPS; (B) LPS-mediated inflammatory induction conditions. Detailed Implementation

[0044] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0045] Hupyridone (10)-Tacrine dimer A10E is synthesized chemically using huperzine A and tacrine as the basic molecular framework. Its English name is Hupyridone(10)-Tacrine, abbreviated as A10E, and its chemical formula is C10. 32 H 44 N4O, molecular formula as follows Figure 1 As shown in Figure A. The A10E used in this invention was synthesized by Chengdu Ruizhi Chemical Research Co., Ltd. based on existing technology (contract number: CD-21060210). A10E is a white crystalline powder with a molecular weight of 500.72 and a purity of ≥99%.

[0046] Polylactic-co-glycolic acid copolymer (50:50) is chemically synthesized from polylactic acid (PLA) and polyglycolic acid (PGA) in a 50:50 ratio, using these as the basic molecular framework. The synthesis technology used is patented. English name: Poly(lactic-co-glycolic acid)(50:50), abbreviated as PLGA(50:50), chemical formula: (C3H6O3.C2H4O3)n, molecular formula as follows: Figure 1 As shown in Figure B. The PLGA (50:50) used in this invention was purchased from Shandong Academy of Pharmaceutical Sciences, CAS No.: 34346-01-5, batch No.: 21071601, molecular weight: 96000, viscosity: 0.71 dL / g.

[0047] Example 1: Chemical Synthesis of A10E

[0048] With the assistance of ChemPartners, the applicant prepared Tacrine(10)-Hupyridone (i.e., A10E). The chemical formula of A10E is C 32 H 44 N4O, with a molecular weight of 500.72, has the following specific synthetic route: Figure 2 As shown. The synthesized A10E uses... 1Molecular assays were performed using ¹H-NMR, and the results were as follows: Figure 3 As shown. Additionally, as... Figure 4 The chromatographic data showed peaks from two different detection methods (DAD and MS). The average peak at 6.02 mins was for the A10E molecule, and the mass spectra showed that M+H, M+2H, and M+3H were at 501.4, 502.4, and 503.4, respectively.

[0049] Based on the Ellman principle, AChE activity was determined. Acetylcholinesterase hydrolyzes acetylcholine to produce choline and acetic acid. Choline then reacts with a thiol-based chromogenic agent to generate a yellow compound called TNB. Colorimetric analysis was performed at 412 nm, and the amount of hydrolysis products indicated acetylcholinesterase activity. This invention compared the IC50 of Donepezil and A10E. Donepezil is a well-established AChE inhibitor with a high IC50 value. 50 =0.016899 μM, which is 22 times that of A10E. This means that A10E is a more effective AChE inhibitor compared to donepezil. Figure 5 The data shows that Donepezil IC50 = 0.0169 μM; A10E IC50 = 0.0169 μM. 50 =0.0008μM.

[0050] Example 2: Fabrication and Characterization of PLGA / A10E Scaffold

[0051] Fabrication process of PLGA / A10E scaffold:

[0052] (1) Weigh 30 mg of huperzine methyl dimer (i.e., A10E), place it in a 50 mL centrifuge tube, and add an appropriate amount of 6 μL of DMSO according to its solubility to dissolve it completely.

[0053] (2) Then weigh 10g of PLGA (50:50) and place it in a 100mL beaker. Add 40mL of 1,4-dioxane and stir for 12 hours. Stir in a closed mechanical stirring at room temperature until the solute is fully dissolved and dispersed to prepare 3D printing "ink" with PLGA (50:50) as the solute.

[0054] (3) Add huperzine methyl dimer A10E before 3D printing and mix thoroughly. After pre-cooling the 3D printer to -30℃, set the printing parameters as follows: size 40mm×40mm×5mm, line spacing 0.45mm, layer height 0.15mm, nozzle movement speed 20.0mm / s; material fill 1.0mm. 3 / s, with a printhead inner diameter of 400μm. After printing, the product is placed in a freeze dryer and freeze-dried under vacuum for 72 hours to remove organic solvents from the material, obtaining a tissue engineering scaffold PLGA / A10E loaded with 0.3% A10E.

[0055] The PLGA / A10E stent was scanned using a Skyscan 1176 micro-CT scanner with the following parameters: voltage: 40 kV, current: 497 μA, resolution: 9 μm, no filter. The pore sizes on the front and sides were analyzed after scanning. Results are as follows: Figure 6 As shown, the front pore diameter is 185.88±25.57μm, the side pore diameter is 232.79±17.00μm, and the porosity is 44%.

[0056] Example 3: Load efficiency and release characteristics of A10E in PLGA / A10E stent

[0057] 1. Load rate detection of A10E in PLGA / A10E bracket:

[0058] (1) Accurately weigh the PLGA / A10E stent, which weighs 106.38 mg. Place the stent in two 5 mL centrifuge tubes, add 1 mL of ethyl acetate to each tube, sonicate for 5 min until the stent is completely dissolved, then add an equal amount of methanol to extract the sample. Vortex for 5 min, then centrifuge at 12000 r for 10 min.

[0059] (2) Take the supernatant, dry it, redissolve it in 500 μL of methanol, filter it through a 0.45 μm filter membrane, and inject the filtrate. The HLPC detection conditions are as follows: mobile phase A: 0.05% formic acid-water; mobile phase B: 0.05% formic acid-acetonitrile; column: Agilent Zorbax Eclipse XDB-C18, 4.6×250 mm, 5 μm; flow rate: 1 mL / min; injection volume: 10 μL; gradient conditions: 0-11 min 5% B-100% B, 11-16 min 100% B; detection wavelength: 254 nm. At the same time, accurately weigh an appropriate amount of A10E and dissolve it in methanol to prepare 2 mL of A10E stock solution with a concentration of 0.5 μg / mL.

[0060] (3) The stock solution was diluted with methanol to obtain standard reference solutions with concentrations of 500, 100, 50, 25, 10, and 5 μg / mL. 10 μL of each solution was injected under the optimized analytical conditions. A linear regression was performed using weighted average (W = 1 / X) with A10E concentration as the x-axis and peak area as the y-axis to obtain the linear equation, standard curve, and calculation formula, as follows: Figure 7 The formula for the well-fitted curve is y = 13966x + 102719 (R²). 2=0.9986).

[0061] This invention calculates the PLGA / A10E loading rate based on the peak area of ​​the tested sample. The results show that the peak area of ​​the tested sample is 3,171,648 μm. Figure 8 Substituting the values ​​into the standard curve, the concentration of the sample to be tested was calculated to be 219.7428756 μg / mL.

[0062] The theoretical drug content of the stent = stent weight × 0.3%, i.e., 106.38 × 0.3% = 0.31914 mg;

[0063] Actual drug content = concentration of the sample to be tested * volumetric volume, i.e. 219.7428756 μg / mL × 500 μL × 10-6 = 0.109871438 mg;

[0064] Loading rate = actual drug content / theoretical drug content × 100%, i.e. 0.109871438 / 0.31914 × 100% = 34.4273478%.

[0065] 2. A10E release characteristics in PLGA / A10E stents:

[0066] Place 300 mg of the stent in 5 mL centrifuge tubes (N=3), and add 3 mL of pH 7.4 PBS (m(g):V(PBS,mL) = 1:10) to each sample vial sequentially. Place the sample vials in a constant temperature shaker, setting the temperature to 37±1℃ and the shaker speed to 70 rpm, according to GB / T 16886.13-2001. After soaking for 24 hours, collect the extract and determine the A10E content in the extract using HPLC. The HLPC detection conditions were as follows: Mobile phase A: 0.05% formic acid-water; Mobile phase B: 0.05% formic acid-acetonitrile; Column: Agilent Zorbax Eclipase XDB-C18, 4.6×250mm, 5μm; Flow rate: 1mL / min; Injection volume: 10μL; Gradient conditions: 0-11min 5%B-100%B, 11-16min 100%B; Detection wavelength: 254nm. Simultaneously, an appropriate amount of A10E was accurately weighed and dissolved in methanol to prepare a 0.5μg / mL A10E stock solution (2mL). The stock solution was then diluted with methanol to obtain standard reference solutions with concentrations of 500, 100, 50, 25, 10, and 5μg / mL. Inject 10 μL of each sample under the optimized analytical conditions. Plot A10E concentration on the x-axis and peak area on the y-axis, and perform a weighted linear regression (W = 1 / X) to obtain the linear equation, standard curve, and calculation formula, as follows. Figure 9 The formula for the well-fitted curve is y = 17935x +

[0067] 105282(R 2 =0.9985).

[0068] This invention calculates the release rate of A10E in the PLGA / A10E stent based on the peak area of ​​the tested samples. The results show that the peak areas of the tested samples are 525154, 583368, and 555474, respectively. Figure 10 .

[0069] Substituting the values ​​into the standard curve, the calculated concentrations of the test samples were 28.69395038, 31.93978255, and 30.38449958 μg / mL.

[0070] The drug content of the stent = stent weight × 0.3% × loading rate, that is, 320.19 × 0.3% × 70.56% = 0.67777819 mg, 321.33 × 0.3% × 70.56% = 0.68019134 mg, 321.10 × 0.3% × 70.56% = 0.67970448 mg;

[0071] Drug release amount = concentration of the sample to be tested * volume, i.e. 28.69395038μg / mL × 3mL = 86.0818511μg, 31.93978255μg / mL × 3mL = 95.81934764μg, 30.38449958μg / mL × 3mL = 91.15349875μg;

[0072] Release rate = Drug release amount / Stent drug content × 100%, i.e., 86.0818511 / 0.67777819 × 10³ × 100% = 12.70%, 95.81934764 / 0.68019134 × 10³ × 100% = 14.09%, 91.15349875 / 0.67970448 × 10³ × 100% = 13.41%. In summary, after 24 hours of in vitro degradation, the release rate of A10E from the stent is approximately 12%.

[0073] Example 4: Effect of PLGA / A10E scaffold in vitro degradation solution on ATDC5 chondrogenic activity under non-inflammatory or inflammatory conditions

[0074] 1. Preparation of stent degradation and release solution:

[0075] (1) The PLGA / A10E scaffold was sterilized by 15 KGy irradiation and in vitro extracted at a rate of 0.1 g / ml (scaffold weight / extract volume). The extract was prepared in MEM medium containing 10% serum and 1% antibiotics. After soaking the scaffold in the extract, it was sealed and shaken at room temperature for 24 hours, with aseptic operation throughout the process. The extract was collected after 24 hours and named the first extract.

[0076] (2) Then add the same volume of MEM medium for extraction and repeat the above operation. After 24 hours, collect the extract and name it the second extract. Filter all extracts with a 0.22 μm filter and store the filtrate at 4 degrees Celsius for later use.

[0077] (3) The concentration of A10E in the extract can be estimated based on the A10E loading rate (34.4%) and the release rate (12%) after soaking for 24 hours obtained in Example 2. The calculation method is as follows:

[0078] One 1cm×1cm×1cm PLGA / A10E stent weighs approximately 0.22g. The A10E content is 0.3%, meaning the mass of A10E in the stent = 0.3% × 0.22g × 34.4% = 0.000337g = 0.227mg. Therefore, the molar mass of A10E = 0.000227g / 500mol / g

[0079] =0.000000454mol=0.454μmol.

[0080] Based on the extraction ratio of 0.1 g / mL: the concentration of A10E after complete extraction of one scaffold is 0.454 μmol / 2.2 ml = 0.2 μm / ml = 200 μM. Therefore, the concentration of A10E in the first extract after 24 hours of extraction is 200 μM * 12% = 24 μM.

[0081] Therefore, theoretically, the A10E concentration in the second immersion solution is 200μM - 24μM * 12% = 21μM. When the first immersion solution is diluted 5 times, 10 times, and 20 times, the A10E concentrations are 4.8μM, 2.4μM, and 1.2μM, respectively; while when the second immersion solution is diluted 5 times, 10 times, and 20 times, the A10E concentrations are 4.2μM, 2.1μM, and 1.05μM, respectively.

[0082] 2. Cell biosafety testing:

[0083] The filtered extract was diluted with MEM medium containing 10% serum and 1% penicillin antibody to obtain extracts with dilution factors of 0, 5, 10, and 20. Cell safety analysis was then performed using the mouse chondrogenic cell line (ATDC5 cells). Specific procedures: ATDC5 cells were cultured in DMEM / F12 medium containing 10% serum and 1% penicillin antibody. Cells were seeded at 0.5 million cells per well in 96-well plates and cultured for 2 days. The culture medium was then replaced with the extract and cultured for 48 hours. The experiment was divided into a normal group, a 0-fold dilution group (0), a 5-fold dilution group (5), a 10-fold dilution group (10), and a 20-fold dilution group (20). At the endpoint, the extract culture medium was removed, and 10% CCK-8 reagent was added to each well for further incubation for 3 hours. The OD value was measured at 490 nm, and the OD values ​​of each group were normalized to the OD value of the normal group.

[0084] The results are as follows Figure 11 As shown, both the first and second extract stock solutions (i.e., the 0-fold dilution group) exhibited significant cytotoxicity. Therefore, this invention screened for 5-fold (5) and 10-fold (10) dilutions for subsequent chondrogenic differentiation experiments.

[0085] The specific procedure was as follows: after diluting the extract 5-fold and 10-fold, 10 ng / ml TGF-β, 10% ITSpremix, 0.1 μM dexamethasone (Dex), 1% sodium pyruvate, and 1 μM vitamin C phosphate were added to prepare chondrogenic differentiation induction solutions. ATDC5 cells were seeded at 5,000 cells per well in 96-well plates and cultured for 2 days. The cultured solution was then replaced with diluted chondrogenic differentiation induction solution containing the extract and cultured for 48 hours. The experiment was divided into a chondrogenic differentiation control group (Control), a 5-fold (5) extract, and a 10-fold (10) extract. At the endpoint, the extract culture medium was removed, and 10% CCK-8 reagent was added to each well for further incubation for 3 hours. The OD value was measured at 490 nm, and the OD values ​​of each group were normalized to the OD value of the Normal group. The results showed that the chondrogenic differentiation induction solution containing the extract could enhance cell activity in the chondrogenic differentiation system.

[0086] 3. Chondrogenic differentiation experiment under non-inflammatory conditions:

[0087] Based on the above results, this invention uses extracts diluted 5 times and 10 times to conduct chondrogenic differentiation experiments.

[0088] The specific procedure was as follows: After diluting the extract 5-fold and 10-fold, 10 ng / ml TGF-β, 10% ITSpremix, 0.1 μM dexamethasone (Dex), 1% sodium pyruvate, and 1 μM vitamin C phosphate were added to prepare chondrogenic differentiation induction solutions. ATDC5 cells were seeded at 100,000 cells per well in 12-well plates and cultured for 2 days. The cultured cells were then replaced with the diluted chondrogenic differentiation induction solution containing the extract and cultured for another 8 days, with the chondrogenic differentiation induction solution being replaced every 2 days. The experiment was divided into a normal growth culture group, a chondrogenic differentiation control group, an A10E (10 μM, 20 μM) group, and 5-fold and 10-fold dilution groups of the extract. At the end of the experiment, the cells were fixed with 4% paraformaldehyde (neutral) for 30 minutes, washed three times with sterile double-distilled water, stained with 1% Alcian Blue staining working solution, and incubated overnight at room temperature in the dark. The next day, the dye solution was discarded, and the sample was washed three times with sterile double-distilled water and photographed under a white light microscope.

[0089] The results are as follows Figure 12 As shown, both the first and second extracts in the Control group, diluted 5-fold and 10-fold, promoted cartilage matrix formation and accelerated the differentiation of ATDC5 cells into chondrocytes. However, huperzine medimer A10E at 10 μM and 20 μM partially inhibited the differentiation of ATDC5 cells into chondrocytes.

[0090] 4. Chondrogenic differentiation experiment under inflammatory conditions:

[0091] In this invention, extracts with a 5-fold and a 10-fold dilution were used to conduct chondrogenic differentiation experiments.

[0092] The specific procedure was as follows: After diluting the extract 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. ATDC5 cells were seeded at 100,000 cells per well in 12-well plates and cultured for 2 days. The cells were then cultured in MEM medium containing 20 ng / ml bacterial endotoxin (LPS) for 1 hour, then the LPS-containing medium was discarded, and the cells were replaced with the diluted chondrogenic differentiation induction solution containing the extract and cultured for another 8 days, changing the chondrogenic differentiation induction solution every 2 days. The experiment was divided into a normal growth culture group, a chondrogenic differentiation control group, an A10E (5 μM) group, a 5-fold dilution group, and a 10-fold dilution group. 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, stained with 1% Alcian Blue staining working solution, and incubated overnight at room temperature in the dark. The next day, the staining solution was discarded, the cells were washed three times with sterile double-distilled water, and photographed under a white light microscope. The results showed ( Figure 13After LPS treatment, normal ATDC5 cells showed significant apoptosis and shedding. In contrast, the control group treated with LPS showed that both the first and second extracts, diluted 5-fold and 10-fold, promoted cartilage matrix formation and accelerated ATDC5 cell differentiation into chondrocytes in the LPS-mediated inflammatory environment. However, the huperzine dimer A10E (5 μM) compound was less effective in promoting chondrogenesis than the PLGA / A10E scaffold degradation dilution (in Example 2, the concentration of A10E after 5-fold dilution was between 4-5 μM).

[0093] Example 5: Effects of PLGA / A10E scaffold in vitro degradation solution on cartilage matrix formation and degradation under non-inflammatory and inflammatory conditions.

[0094] In this invention, the first extract was diluted 10 times before chondrogenic differentiation experiments were conducted.

[0095] The specific procedure was as follows: After the first 10-fold dilution of the extract, 10 ng / ml TGF-β, 10% ITSpremix, 0.1 μM dexamethasone (Dex), 1% sodium pyruvate, and 1 μM vitamin C phosphate were added to prepare chondrogenic differentiation induction solutions. ATDC5 cells were seeded at 300,000 cells per well in 6-well plates and cultured for 2 days. Cells were then cultured for 1 hour in MEM medium with and without 20 ng / ml bacterial endotoxin (LPS). The medium was then discarded, and the cells were cultured again for 6 days in diluted chondrogenic differentiation induction solution containing the extract. The chondrogenic differentiation induction solution was changed every 2 days. Under non-inflammatory conditions, the experiment was divided into a normal growth culture group (Normal), a chondrogenic differentiation control group (Control), a PLGA scaffold extract group (PLGA), and a PLGA / A10E scaffold extract group (PLGA / A10E). The LPS-mediated inflammatory experiment was divided into three groups: a chondrogenic differentiation control group (LPS+Control), a PLGA scaffold extract release group (LPS+PLGA), and a PLGA / A10E scaffold extract release group (LPS+PLGA / A10E). At the end of the experiment, the culture medium was discarded, and the cells were washed with cold PBS. 300 μL of RIPA protein lysis buffer was added to each well, and the cells were lysed on ice for 30 minutes. The lysate was collected and centrifuged at 8000 rpm for 30 minutes at 4°C. The supernatant of the cell protein solution was collected, and the protein concentration was determined using the BCA method. Loading buffer was then added and the protein was dissolved in boiling water for 10 minutes. After cooling, Western blotting was performed. The protein loading volume was 30 μg, using an 8% separating gel at 120V (20mA) for 70 minutes. Transfer was then performed at 300mA for 90 minutes. Sox9 antibody (Abcam, ab185966, dilution 1:2000), MMP-13 antibody (Abcam, ab39012, dilution 1:2000), and GAPDH antibody (Abcam, ab8245, dilution 1:5000) were incubated overnight at 4°C, followed by incubation of secondary antibody (1:10000) at room temperature for 2 hours. After cleaning, the samples were developed, observed, and photographed.

[0096] The results are as follows Figure 14 As shown, under non-inflammatory conditions, PLGA / A10E extract upregulated Sox9 expression. However, under inflammatory conditions, PLGA / A10E extract downregulated MMP-13 expression. Sox9 is a chondrocyte differentiation transcription factor, while MMP-13 is a major cartilage matrix degradation enzyme. In conclusion, PLGA / A10E extract can promote chondrocyte differentiation and inhibit cartilage matrix degradation under inflammatory conditions.

Claims

1. An anti-inflammatory cartilage regeneration material, characterized in that, The anti-inflammatory and cartilage regeneration material is a PLGA / huperzine A derivative dimer scaffold, which contains PLGA (50:50) polymer material and huperzine A derivative dimers loaded on it by 3D printing. The mass ratio of the PLGA to the huperzine A derivative dimer was 10 g: 30 mg. The huperzine A derivative dimer is A10E, which is chemically synthesized using huperzine A and tacardine as the basic molecular framework. A10E has the chemical formula C32H44N4O and the molecular formula is as follows: ； The preparation method of the anti-inflammatory and cartilage regeneration material includes the following steps: (1) Weigh the huperzine A derivative dimer and add dimethyl sulfoxide to dissolve it. The mass-to-volume ratio of the huperzine A derivative dimer to dimethyl sulfoxide is 30 mg: 6 μL. (2) Weigh PLGA (50:50), add 1,4-dioxane, stir for 12 hours, add the solution obtained in step (1), mix evenly, the mass and volume ratio of PLGA (50:50) to 1,4-dioxane is 10 g:40 mL; (3) 3D printing is performed. After completion, the product is obtained and placed in a freeze dryer for vacuum drying for 72 hours to obtain an anti-inflammatory cartilage regeneration material, namely PLGA / huperzine A derivative dimer scaffold. The 3D printing conditions described in step (3) are as follows: After pre-cooling the 3D printer to -30℃, 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 moving speed 20.0 mm / s; material filling 1.0 mm. 3 / s, printhead inner diameter 400 μm; In step (2), the stirring method is: mechanical stirring in a closed system at room temperature until the solute is fully dissolved and dispersed.

2. The method for preparing an anti-inflammatory cartilage regeneration material as described in claim 1, characterized in that, Includes the following steps: (1) Weigh the huperzine A derivative dimer and add dimethyl sulfoxide to dissolve it. The mass-to-volume ratio of the huperzine A derivative dimer to dimethyl sulfoxide is 30 mg: 6 μL. (2) Weigh PLGA (50:50), add 1,4-dioxane, stir for 12 hours, add the solution obtained in step (1), mix evenly, the mass and volume ratio of PLGA (50:50) to 1,4-dioxane is 10 g:40 mL; (3) 3D printing is performed. After completion, the product is obtained and placed in a freeze dryer for vacuum drying for 72 hours to obtain an anti-inflammatory cartilage regeneration material, namely PLGA / huperzine A derivative dimer scaffold. The 3D printing conditions described in step (3) are as follows: After pre-cooling the 3D printer to -30℃, 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 moving speed 20.0 mm / s; material filling 1.0 mm. 3 / s, printhead inner diameter 400 μm; In step (2), the stirring method is: mechanical stirring in a closed system at room temperature until the solute is fully dissolved and dispersed.

3. The use of the anti-inflammatory and cartilage regeneration-promoting material as described in claim 1 in the preparation of anti-inflammatory, cartilage-protecting, or cartilage-regenerating drugs.

4. The use as described in claim 3, characterized in that, The anti-inflammatory and cartilage regeneration material can promote the differentiation of cartilage precursor cells into cartilage and increase the formation of cartilage collagen matrix and cartilage proteoglycans.

5. The use as described in claim 3, characterized in that, The anti-inflammatory and cartilage regeneration-promoting material promotes SOX9 expression under non-inflammatory conditions.

6. The use as described in claim 3, characterized in that, The anti-inflammatory and cartilage regeneration-promoting material can inhibit the expression of MMP-13 under LPS-mediated inflammatory conditions, thereby promoting cartilage matrix synthesis and inhibiting cartilage matrix degradation.

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