Use of astragaloside in preparation of medicine for promoting BMSCs osteogenic differentiation of patients with congenital scoliosis

Abstract

The present application relates to a new medical use of Astragaloside IV, and particularly relates to the use of Astragaloside IV in the preparation of a medicine for promoting the osteogenic differentiation of BMSCs of a patient with congenital scoliosis. The present application discloses that Astragaloside IV can specifically up-regulate the expression of WISP2, and then activate the Wnt / β-catenin pathway, significantly improve the alkaline phosphatase activity, mineralization capacity and osteogenic related gene expression of CS-BMSCs. Through cell induction, animal implantation and targeted sustained-release preparation and other experimental means, the present application confirms the safety and effectiveness of Astragaloside IV in repairing CS-related osteoporosis, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to novel pharmaceutical uses of astragaloside A, and more particularly to the use of astragaloside A in the preparation of a drug that promotes osteogenic differentiation of bone mesenchymal stem cells (BMSCs) in patients with congenital scoliosis. Background Technology

[0002] Congenital scoliosis (CS) is a three-dimensional deformity caused by disruption of vertebral segmentation or formation during the 4th–6th week of embryonic development. Its incidence rate is approximately 0.5‰–1‰ both domestically and internationally, with over 60% of cases accompanied by abnormalities in the heart, kidneys, thoracic cage, or neural axis. In addition to physical deformities and progressive imbalances, clinical imaging and bone mineral density-dual-energy X-ray absorptiometry (DXA) studies show that CS patients often have insufficient bone mineralization or even osteoporosis, with features such as thinning and decreased mechanical strength in the trabeculae of the lumbar spine, femoral neck, and iliac crest. Microscopic and molecular evidence suggests that one of the root causes of pathological bone turnover is impaired osteogenic differentiation potential of bone marrow mesenchymal stem cells (BMSCs); however, this aspect has long been overlooked in CS.

[0003] Bone mesenchymal stem cells (BMSCs) are oriented towards the osteogenic lineage through signaling networks such as Wnt / β-catenin, serving as "seed" cells for bone remodeling in adults and bone growth in children. β-catenin deficiency drives BMSCs to favor the cartilage or adipose lineage, leading to bone loss; conversely, β-catenin nuclear translocation can upregulate core bone-forming transcription factors such as RUNX2 and OSX. Recent studies have demonstrated that the branched-chain protein WNT-1-inducible signaling pathway protein-2 (WISP2, also known as CCN5) in the Wnt pathway plays a "signal amplifier" role in this process—WISP2 can synergistically inhibit β-catenin degradation and promote bone matrix mineralization with the LRP5 / 6 complex.

[0004] Currently, surgical correction (in situ fusion, hemivertebrae osteotomy, growth rods, VEPTR, etc.) is the mainstream approach to control the progression of osteoporosis, but it does not directly improve bone quality itself. Postoperative complications such as delayed bone graft fusion, loosening or breakage of internal fixation still exist, suggesting that the bone biological environment must be optimized simultaneously. For osteoporosis associated with CS, bisphosphonates, PTH analogs, or calcium-vitamin D supportive therapy are commonly used, but these drugs are not designed to target the BMSCs-WISP2-Wnt pathway, resulting in limited efficacy and adverse reactions such as hypocalcemia and adenoma risk. Meanwhile, gene or protein replacement strategies targeting BMSCs (such as viral overexpression of WISP2), although feasible at the experimental level, are limited by vector safety, transfection efficiency, and the difficulty of clinical application, making it difficult to become a routine standard in the short term.

[0005] Astragalus is a traditional Chinese medicine for "tonifying qi and strengthening the exterior, supporting the interior and promoting tissue regeneration," and its main saponin monomer is astragaloside A (AST). Modern pharmacology shows that AST has multiple activities, including antioxidant, angiogenesis, and immunomodulatory effects. Studies in the field of bone have shown that AST can enhance ALP activity and promote the formation of mineralized nodules in models such as periodontal ligament stem cells, osteogenic precursor MC3T3-E1, and BMSCs, accompanied by activation of the PI3K / Akt or Wnt / β-catenin pathways.

[0006] Chinese invention patents (such as CN117064902A and CN104095992A) have proposed "the application of AST-containing compositions in the repair of osteoporosis, osteoarthritis or dental diseases". It can be seen that although the osteogenic potential of AST has been preliminarily affirmed, its use in targeting WISP2 regulation and serving the low bone density of CS patients is a blank area. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention utilizes the positive regulatory effect of astragaloside A on WISP2 and proposes its application in the preparation of drugs that promote osteogenic differentiation of bone mesenchymal stem cells (BMSCs) in patients with congenital scoliosis. This provides a new technical solution for bone metabolic reconstruction in CS patients and has significant innovation and clinical application value.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The use of astragaloside IV (AST-IV) in the preparation of a drug that promotes osteogenic differentiation of bone mesenchymal stem cells (BMSCs) from patients with congenital scoliosis, wherein the drug upregulates the expression of WNT-1-induced signaling pathway protein 2 (WISP2) and activates the Wnt / β-catenin signaling pathway; and is used to increase alkaline phosphatase activity, mineralized nodule formation, and expression levels of osteogenic-related genes RUNX2, COL1A1, OSX, OPN, and OCN in BMSCs from patients with congenital scoliosis.

[0010] Preferably, the drug is a solution, lyophilized powder, or polylactic acid-hydroxyapatite nanosphere sustained-release formulation containing 1.0 mg / mL to 10 mg / mL astragaloside A.

[0011] Preferably, the drug further comprises a pharmaceutically acceptable carrier and 1-10% (w / w) of a stabilizer, oligosaccharide, or surfactant.

[0012] Preferably, the drug is administered via subcutaneous injection or local intraosseous injection, with a single dose of 0.05 mg / kg to 2 mg / kg body weight, administered once every 5 to 10 days for 4 to 12 weeks.

[0013] Preferably, the drug is suitable for the treatment or prevention of osteoporosis or insufficient bone mineralization in patients aged 4 to 25 years with congenital scoliosis, Cobb angle ≥10°, and DXA T value ≤-2.0SD.

[0014] Preferably, the drug is exposed to BMSCs from patients with congenital scoliosis at a final concentration of 4 µg / mL to 16 µg / mL in vitro for 7 to 21 days, thereby increasing the expression of WISP2 and β-catenin mRNA by at least 1.5 times.

[0015] Preferably, the drug is used in combination with 10–100 ng / mL fibroblast growth factor-2 (FGF-2) or bone morphogenetic protein-2 (BMP-2) to synergistically enhance osteogenic differentiation of CS-BMSCs.

[0016] Preferably, the drug is a pH-sensitive or esterase-degradable microsphere formulation with a particle size of 100 nm to 500 nm, and it releases more than 80% of astragaloside A within 72 hours.

[0017] Furthermore, the present invention also discloses a pharmaceutical composition for promoting osteogenic differentiation of BMSCs in patients with congenital scoliosis. The pharmaceutical composition is a solution, lyophilized powder, or polylactic acid-hydroxyapatite nanosphere sustained-release formulation containing 1.0 mg / mL to 10 mg / mL astragaloside.

[0018] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and 1–10% (w / w) of a stabilizer, oligosaccharide, or surfactant; and / or, the pharmaceutical composition further comprises 10–100 ng / mL of fibroblast growth factor-2 (FGF-2) or bone morphogenetic protein-2 (BMP-2).

[0019] Due to the adoption of the above-mentioned technical solutions, this invention has achieved significant and comprehensive technical effects in terms of molecular target accuracy, osteogenic enhancement, safe applicable population, and delivery method, as detailed below:

[0020] 1. Precise Targeting: This invention demonstrates for the first time that astragaloside A (AST) can significantly upregulate WISP2 in bone marrow mesenchymal stem cells (BMSCs) of CS patients, and sequentially activate the classical Wnt / β-catenin signaling pathway. This effect specifically targets the low WISP2 expression phenotype unique to CS-BMSCs, representing a "deficiency-dependent" compensation, thus avoiding the off-target risks that may result from broad-spectrum activation of the Wnt pathway.

[0021] 2. Significant osteogenic enhancement: Within the range of 4–16 µg / mL, AST in this invention increases ALP activity, calcified nodule area, and bone markers such as RUNX2, COL1A1, and OCN by 60–90%. After AST-preinduced CS-BMSCs were implanted into bone defects in animals, bone volume / tissue volume (BV / TV) increased by approximately 45% and compressive strength increased by approximately 38% after 8 weeks, showing no significant difference compared to autologous bone transplantation.

[0022] 3. Wide safety window: AST promotes cell proliferation and differentiation at ≤16µg / mL; the inhibitory effect at ≥32µg / mL provides a clear safety threshold for the upper dose limit. Compared with viral vectors or recombinant proteins, AST has low immunogenicity and no risks of integration, ectopic ossification, or hypercalcemia.

[0023] 4. Delivery-friendly: This invention uses PLGA-hydroxyapatite nanospheres for encapsulation, allowing AST to be continuously released within the defect microenvironment for more than 72 hours. A single local administration can cover the osteogenic window of one week, significantly reducing the frequency of clinical operations.

[0024] 5. Comprehensive advantages comparison: Compared with traditional bisphosphonates, PTH or BMP-2, this invention has the triple advantages of high specificity, low side effects and controllable cost. The osteogenic enhancement is comparable to gene knock-in-WISP2, but avoids the regulatory and safety obstacles of viral and protein drugs.

[0025] Overall, this invention innovatively constructs a complete drug-signal-cell pathway from AST to WISP2 to Wnt / β-catenin to osteogenic bone formation, achieving clinically feasible and precise intervention in repairing osteocyte-associated bone hypoplasia. It provides a safe, efficient, and easily industrialized new solution for bone metabolic remodeling in patients with refractory spinal deformities. Attached Figure Description

[0026] Figure 1 Microscopic morphological observation of BMSCs (×10); among which Figure 1 A: cultured for 2 days, B: cultured for 4 days, C: cultured for 10 days.

[0027] Figure 2 Image showing the results of surface-labeled biochemical cytometry identification of human BMSCs.

[0028] Figure 3 .Staining images of BMSCs after osteogenic induction; in which Figure 3 (A) ALP staining, (B) ARS staining, (C) Alcian blue staining after chondrogenic induction, (D) Oil Red O staining after adipogenic induction.

[0029] Figure 4 ALP staining and activity assay diagram.

[0030] Figure 5 ARS staining and quantitative analysis of calcium nodules.

[0031] Figure 6 .RT-qPCR detection of osteogenic-related markers and WISP2 mRNA expression levels.

[0032] Figure 7 Western blotting analysis of osteogenic markers and WISP2 protein expression levels.

[0033] Figure 8 The graph shows the changes in WISP2 mRNA expression level detected by RT-qPCR after transfection, and the changes in WISP2 protein expression level detected by Western Blot.

[0034] Figure 9 ALP staining and ALP activity assay (siWISP2 represents WISP2 knockdown).

[0035] Figure 10 .ARS staining and quantitative analysis of calcium nodules (siWISP2 represents WISP2 knockdown).

[0036] Figure 11 .RT-qPCR detection of mRNA expression levels of COL1A1, RUNX2, OSX, OPN, and OCN (siWISP2 represents WISP2 knockdown).

[0037] Figure 12 Western blot plot showing protein expression levels of RUNX2, OPN, and OCN (siWISP2 represents WISP2 knockdown).

[0038] Figure 13 ALP staining and ALP activity assay (lenti-WISP2 represents WISP2 overexpression).

[0039] Figure 14 ARS staining and quantitative analysis of calcium nodules (lenti-WISP2 represents WISP2 overexpression).

[0040] Figure 15 RT-qPCR detection of mRNA expression levels of COL1A1, RUNX2, OSX, OPN, and OCN (lenti-WISP2 represents WISP2 overexpression).

[0041] Figure 16 Western blot plot showing protein expression levels of COL1A1, RUNX2, OSX, OPN, and OCN (lenti-WISP2 represents WISP2 overexpression).

[0042] Figure 17 Effect of different concentrations of AST on the proliferation of CS-BMSCs at different time points as detected by CCK-8 assay.

[0043] Figure 18 ALP staining and ALP activity assay diagram.

[0044] Figure 19 ARS staining and quantitative analysis of calcium nodules.

[0045] Figure 20 .RT-qPCR detection of mRNA expression levels of COL1A1, RUNX2, OSX, OPN, OCN and WISP2.

[0046] Figure 21 .WB detection of protein expression levels of RUNX2, OPN, OCN and WISP2.

[0047] Figure 22 A schematic diagram of the molecular docking results between AST and WISP2 protein.

[0048] Figure 23 ALP staining and ALP activity assay diagram.

[0049] Figure 24 ARS staining and quantitative analysis of calcium nodules.

[0050] Figure 25 RT-qPCR was used to detect the mRNA of β-catenin, COL1A1, RUNX2, OSX, OPN, OCN, and WISP2.

[0051] Figure 26 .Western Blot analysis of protein expression levels of β-catenin, RUNX2, OPN, and WISP2. Detailed Implementation

[0052] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of the present invention.

[0053] Example 1: Study on the sorting, culture, and osteogenic differentiation induction of BMSCs from CS patients and normal individuals.

[0054] I. Materials and Methods

[0055] (a) Experimental materials

[0056] 1. Experimental Samples

[0057] The sample consisted of CS patients undergoing inpatient surgery and healthy subjects. Inclusion criteria for the CS group were: 1. Diagnosis of CS via clinical examination, physical examination, and imaging (spine X-ray / CT / MRI); 2. Complete clinical and imaging data; 3. Bone mineral density (DEXA) showing osteopenia or osteoporosis. Inclusion criteria for the control group were: 1. Complete clinical and imaging data; 2. No spinal or other skeletal developmental abnormalities; 3. Normal bone mineral density results. This study was reviewed by the Ethics Committee of Hangzhou Xiaoshan District Traditional Chinese Medicine Hospital.

[0058] 2. Experimental equipment

[0059] Table 1-1 Main Experimental Equipment

[0060]

[0061] 3. Experimental reagents

[0062] Table 1-2 Main Experimental Reagents

[0063]

[0064] (II) Experimental Methods

[0065] 1. Isolation and culture of BMSCs

[0066] 1.1 Isolation of BMSCs

[0067] 5 ml of sterile bone marrow obtained clinically was placed in an ice box and quickly transferred to the laboratory. 40 ml of erythrocyte lysis buffer was added and allowed to stand for 10-15 minutes until the solution became clear. The solution was then centrifuged at 200 g for 5 minutes, the supernatant was discarded, and 5 ml of complete culture medium (DMEM / F12+FBS+L-Glutamine+double antibiotics) was added. The mixture was then pipetted and seeded into T-25 culture flasks and placed in a 5% CO2, 37°C incubator. After 24 hours, the medium was changed to remove any non-adherent cells. The medium was then changed every two days thereafter.

[0068] 1.2 Passage of BMSCs

[0069] When the cell confluence reaches 70%-80% as observed under a microscope, the cells are passaged. The culture medium is aspirated, and the cells are washed twice with PBS. Tryple enzyme is added and the cells are digested in an incubator for 5 minutes. An equal volume of complete culture medium is then added to stop the digestion. The cell suspension is then transferred to a 15 ml sterile centrifuge tube and centrifuged at 500 g for 5 minutes. The supernatant is removed, and the cell pellet is resuspended in complete culture medium. The cells are then passaged into new culture flasks at a 1:3 inoculation ratio and finally placed in a CO2 cell culture incubator.

[0070] 1.3 Cryopreservation of BMSCs

[0071] Digest and centrifuge the cells according to steps 1.2, remove the supernatant, add cell freezing solution to resuspend the cell pellet, aliquot into cryovials, label them, and store them in a -80°C freezer.

[0072] 1.4 Recovery of BMSCs

[0073] The frozen cells were quickly transferred to a 37°C water bath and thawed. After thawing, they were transferred to sterile centrifuge tubes and centrifuged at 500g for 5 minutes. The supernatant was discarded, and the cells were resuspended in complete culture medium. The cells were then seeded into culture flasks and placed in a cell culture incubator for incubation.

[0074] 2. Flow cytometry identification of BMSCs

[0075] The trypsin-digested BMSCs suspension was transferred to a 1.5 mL sterile centrifuge tube and centrifuged at 500g for 5 min to collect the cell pellet. The cells were resuspended in pre-chilled PBS (containing 1% BSA) at a cell density of 1×10⁻⁶ cells / mL. 6 Cells / mL; 100 μl of cell suspension was aliquoted into EP tubes, and primary antibody was added and mixed. The mixture was incubated at 4°C in the dark for 30 min. 1 ml of flow cytometry buffer was added, and the mixture was centrifuged at 250 g for 5 min at 4°C. The cells were washed twice to remove non-specific binding. The supernatant was discarded, and 100 μl of secondary antibody was added. The mixture was incubated again at 4°C in the dark for 30 min. After centrifugation and washing under the same conditions, 3 μl of 7-7-AAD was added, and the mixture was reacted in the dark for 10 min. The cells were then filtered through a 70 μm sterile cell filter and transferred to flow cytometry tubes for analysis. Flow cytometry was used for detection, capturing at least 10 cells / mL per sample. 4 In a live cell event, cells with fluorescence intensity higher than the 95th percentile of the control were identified as the positive population.

[0076] 3. Trilineage-induced differentiation of BMSCs

[0077] 3.1 Osteogenic induction differentiation and staining

[0078] Coat the well plates with 0.1% gelatin overnight before inoculation, then discard the gelatin and dry them in an incubator. Digest and centrifuge the BMSCs according to the above steps, then incubate at 5×10⁻⁶. 3 pcs / cm 2 The cells were seeded at a density of 70-80% in well plates. When the cells reached 70-80% confluence, they were washed with PBS and then added to osteogenic induction differentiation medium (DMEM / F12+FBS+β-glycerophosphate sodium+dexamethasone+ascorbic acid+double antibiotics). The cells were cultured in a 5% CO2, 37°C incubator with the medium changed every 3 days. After 2-4 weeks of induced differentiation, ALP and ARS staining were performed.

[0079] (1) ALP staining

[0080] BMSCs were stained 14 days after osteogenic induction. First, the working solution was prepared by adding 20 μl of 150x NBT solution and 30 μl of 100x BCIP solution to every 3 ml of alkaline phosphatase chromogenic reaction buffer. The osteogenic induction differentiation medium was aspirated, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 2 min, the fixative was aspirated, the cells were washed three times with PBS, and the chromogenic working solution was added and incubated at room temperature in the dark for 30 min. The cells were then observed and photographed under a microscope.

[0081] (2) ALP activity assay

[0082] ①After osteogenic induction of BMSCs for 14 days, the cells were digested and collected into 1.5ml centrifuge tubes, lysis buffer was added and the cells were lysed on ice for 30min. After centrifugation, the supernatant was transferred to a new centrifuge tube.

[0083] ② Chromogenic substrate solution: Dissolve the chromogenic substrate in 2.5 ml of detection buffer and mix well. Keep on ice for later use. Preparation of standard working solution: Dilute 10 μl of 10 mM p-nitrophenol to 200 μl (final concentration 0.5 mM).

[0084] ③ Set up blank control wells, standard wells and sample wells. Add 50 μl of chromogenic substrate solution and 50 μl of detection buffer to the control wells. Add 4, 8, 16, 24, 32 and 40 μl of working solution to the standard wells respectively. Then add detection buffer to make the final volume of each well 100 μl. Add 50 μl of chromogenic substrate solution and 50 μl of sample to the sample wells.

[0085] ④ Mix well by blowing and then incubate in a 37°C incubator for 10 minutes;

[0086] ⑤ Add 100 μl of reaction stop solution to each well to terminate the reaction;

[0087] ⑥ Measure the absorbance at 405 nm using an ELISA reader.

[0088] (3) ARS staining

[0089] BMSCs were stained 21 days after osteogenic induction. The culture medium was removed, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 30 min, washed three times with PBS, added ARS staining solution for 30 min, washed three times with distilled water, and then observed and photographed.

[0090] (4) Quantitative analysis of mineralized nodules

[0091] After acquiring images of mineralized nodules, the culture plate was dried. 1 ml of 10% hexadecylpyridine chloride was added quantitatively to each staining well, and the plate was slowly shaken on a shaker for 20 min until the alizarin red-S dye was completely dissociated from the calcium deposits. Then, 100 μl of the solution was transferred to a 96-well plate, and the absorbance was measured at 570 nm using a microplate reader.

[0092] 3.2 Chondrogenic differentiation and staining

[0093] Coat the well plates with 0.1% gelatin overnight before inoculation, then discard the gelatin and dry them in an incubator. Digest and centrifuge the BMSCs according to the above steps, then incubate at 5×10⁻⁶. 3 pcs / cm 2 The cells were seeded at a density of 80-90% in well plates. After washing with PBS, the cells were added to the prepared complete chondrogenic differentiation induction medium (chondrogenic differentiation basal medium + dexamethasone + ascorbic acid + sodium pyruvate + L-proline + ITS supplement), and cultured in a 5% CO2, 37°C incubator. The medium was changed every 3 days for 4 weeks.

[0094] Alicin Blue Staining: Remove the chondrogenic differentiation-inducing medium, wash 3 times with PBS, then add 4% paraformaldehyde for 30 min; wash 3 times with PBS, then add Alicin Blue staining solution for 20 min; rinse 3 times with PBS, and observe and photograph under a microscope.

[0095] 3.3 Adipogenic induction differentiation and staining

[0096] Coat the well plates with 0.1% gelatin overnight before inoculation, then discard the gelatin and dry them in an incubator. Digest and centrifuge the BMSCs according to the above steps, then incubate at 5×10⁻⁶. 3 pcs / cm 2 The cells were seeded at a density of 100% in well plates. When the cell confluence reached 100%, the culture medium was aspirated, and the cells were washed with PBS and then added to adipogenic differentiation induction medium A (adipogenic differentiation basal medium + special serum + dexamethasone + insulin + IBMX + rosiglitazone + penicillin). The cells were incubated in a 5% CO2, 37°C incubator. After 3 days, the medium was replaced with adipogenic differentiation induction medium B (adipogenic differentiation basal medium + special serum + insulin + penicillin). The "A3B1" alternating induction mode was used, and after 4 cycles, the cells were transferred to medium B for maintenance culture for 4 weeks.

[0097] Oil Red O staining: After removing the culture medium, rinse three times with PBS and fix with 4% paraformaldehyde for 30 min. After washing with PBS again, stain with Oil Red O for 30 min. After staining, wash with PBS and finally observe and take pictures.

[0098] 4. Total RNA extraction and reverse transcription from cells

[0099] 4.1 RNA extraction

[0100] (1) Seed BMSCs in 6-well plates. When the cell confluence reaches 70-80%, replace with osteogenic induction differentiation complete medium for 7-14 days.

[0101] (2) After the cells have been induced, wash them three times with PBS, add 500 μl of Lysis Buffer, and transfer them to an enzyme-free 1.5 ml centrifuge tube by repeatedly pipetting and aspirating.

[0102] (3) Add 500 μl of anhydrous ethanol to the centrifuge tube, mix well by pipetting, and then add it to the RNA column. Centrifuge at 12000g for 1 min.

[0103] (4) After centrifugation, discard the waste liquid, add Wash Buffer to the centrifuge column, centrifuge at 12000g for 1 min, discard the waste liquid, put the RNA column into a new centrifuge tube, and open the cap for 2 min.

[0104] (5) Add 20 μl of Elution Buffer to the column and centrifuge after 2 minutes. Add the solution back to the column and centrifuge again after 5 minutes. Discard the RNA column. The solution at the bottom of the centrifuge tube is the extracted RNA.

[0105] (6) Measure the concentration and purity of the eluted RNA, and label the sample name, time, operator and other information before storing it in a -80℃ refrigerator for later use.

[0106] 4.2 Reverse transcription

[0107] (1) Add 15 μl RNase-free H2O To, 3 μl 5×g DNA digester Mix, and 2 μg total RNA to a 200 μl enzyme-free centrifuge tube, then place it in a 42℃ metal bath for 2 min after short-term centrifugation.

[0108] (7) After the metal bath, add 5 μl of 4×Hifair® Ⅲ Super Mix, mix well, and run reverse transcription.

[0109] 5. RT-qPCR

[0110] (1) Prepare a 10 μl RT-qPCR reaction system

[0111] Table 1-3 RT-qPCR reaction system

[0112]

[0113] (2) Add the prepared solution to the 384 plate, then run it on the instrument. Activate at 50℃ for 2 min; pre-denature at 95℃ for 2 min; anneal at 60℃ for 15 sec; extend at 72℃ for 1 min, and repeat 40 times.

[0114] (2) After the reaction is complete, apply 2 -ΔΔCt The algorithm calculates the fold change in expression between groups. ΔCt = Ct(target) - Ct(GAPDH); ΔCt = ΔCt(experimental) - ΔCt(control), with GAPDH used as an internal control.

[0115] (3) PCR primer sequences

[0116] Table 1-4 RT-qPCR Primer Sequences

[0117]

[0118] 6. Total cellular protein extraction and Western blotting.

[0119] 6.1 Extraction of total cellular protein

[0120] (1) Seed BMSCs in 6-well plates. When the cell confluence reaches 70-80%, replace with osteogenic induction differentiation complete medium for 7-14 days.

[0121] (2) Digest the cells according to step 1.2 to obtain cell precipitate;

[0122] (3) Discard the supernatant, add 300 μl of the prepared RIPA lysis buffer (1 ml RIPA is mixed with 10 μl 100X PMSF and 10 μl protein phosphatase inhibitor), and mix by pipetting. Place on ice for lysis for 30 min.

[0123] (4) After lysis, sonicate to increase protein dissolution, then centrifuge at 12000g for 10 min at 4°C, transfer the supernatant to a new 1.5ml centrifuge tube and store it on ice.

[0124] 6.2 Determination of protein concentration by BCA method

[0125] (1) Dissolve 20 mg BSA in 0.8 ml of the prepared solution (final concentration 25 mg / ml), dispense and dilute to 0.5 mg / ml;

[0126] (2) Prepare the working solution by mixing BCA reagents A and B at a volume ratio of 50:1;

[0127] (3) Dilute the 0.5 mg / ml standard to 20 μl / well using different volumes of diluent and add the solution to a 96-well plate to form standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml.

[0128] (4) Add the protein solution to be tested and 200 μl of BCA working solution to the sample wells and incubate at 37°C for 30 min;

[0129] (5) Read the absorbance at 562 nm using an ELISA reader;

[0130] (6) Plot a standard curve and calculate the protein concentration of the sample;

[0131] (7) Add protein loading buffer (1:4) to the sample with the determined concentration, mix well and transfer to a 99℃ metal bath for denaturation for 10 minutes. After the denaturation is complete, place it in a -80℃ refrigerator for later use.

[0132] 6.3 Western Blot

[0133] (1) Fix the gel plate in the electrophoresis tank, pour in the electrophoresis solution until it covers the sample wells, slowly and vertically pull out the comb, add the protein to the sample wells according to the calculated protein volume, and leave one well on each side for adding markers.

[0134] (2) Turn on the power supply, first set the voltage to 80V, electrophoresis for 30 minutes, then change it to 120V until the strips reach the bottom of the separating gel, then stop electrophoresis;

[0135] (3) Measure and cut the PVDF membrane according to the size of the gel, activate it with methanol for 60 seconds and set it aside. Remove the gel and put it into the transfer solution.

[0136] (4) Construct the transfer membrane interlayer in the transfer solution in sequence: sponge pad → 3 layers of filter paper → adhesive strip → PVDF membrane → 3 layers of filter paper → sponge pad. Place the interlayer into the transfer tank, pour in the transfer solution, and transfer the membrane at a constant current of 400mA for 1-2 hours.

[0137] (5) After the transfer is complete, remove the PVDF membrane, wash it with TBST 3 times for 5 minutes each time, then add QuickBlock™ Western blocking solution and block it on a horizontal shaker for 10 minutes.

[0138] (6) After blocking, wash the membrane 3 times with TBST for 5 minutes each time. Cut the target protein band according to the protein marker position, then put it into the antibody incubation box, add the prepared primary antibody, and then put it on a shaker in a 4°C refrigerator overnight.

[0139] (7) Recover the primary antibody, wash the membrane 3 times with TBST for 5 min each time, then add the secondary antibody and incubate on a shaker for 1 h;

[0140] (8) Recover the secondary antibody, wash the membrane 3 times with TBST for 5 minutes each time, prepare ECL developing solution, place the membrane in a protein scanning membrane instrument, add developing solution to cover the entire membrane, scan and develop, and then use Image J for quantitative analysis.

[0141] 7. Statistical Analysis

[0142] All data are expressed as mean ± standard deviation. Statistical analysis and graphing were performed using GraphPad Prism 10.1.2 software. Comparisons between two groups were conducted using... t The test can be either the Mann-Whitney test or a similar test. For comparisons of multiple groups, one-way ANOVA or the Kruskal-Wallis test can be used. P < 0.05 is statistically significant (*) p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001).

[0143] II. Experimental Results

[0144] 1. Culture and morphological observation of BMSCs

[0145] Primary BMSCs adhered to the culture vessel after 2 days and exhibited uniform morphology and clear edges. After 4 days, they further extended into typical long spindle or fusiform shapes. After 10 days, the cells interconnected and were tightly packed (see...). Figure 1 ).

[0146] 2. Detection of surface antigens in BMSCs

[0147] Flow cytometry analysis revealed that the expression of surface antigens on human BMSCs was highly expressed (≥95%) by typical positive markers CD29, CD105, and CD73 on the cell surface, while the expression of surface markers CD11b, CD34, and CD45 on monocytes, hematopoietic stem cells, and leukocytes was low (≤2%), consistent with the specificity of human BMSC surface antigens. Figure 2 ).

[0148] 3. Results of trilineage differentiation induction of BMSCs

[0149] Osteogenic differentiation: After osteogenic induction, both ALP and ARS staining were positive; Adipogenic differentiation: After adipogenic induction, Oil Red O staining showed significant intracellular lipid droplet aggregation; Chondrogenic differentiation: Alcian blue staining was positive. These results collectively confirm that cultured BMSCs possess trilineage differentiation potential. Figure 3 ).

[0150] 4. Staining results of bone marrow mesenchymal stem cells (BMSCs) after osteogenic induction in normal individuals and CS patients

[0151] Compared with the control group, ALP staining was significantly weakened and ALP activity was reduced in the CS group. Figure 4 ARS staining was lighter, and the number of mineralized nodules was reduced. Figure 5 ).

[0152] 5. RT-qPCR results of osteogenic induction of BMSCs in normal individuals and CS patients

[0153] After osteogenic induction, the mRNA expression levels of osteogenic factors and WISP2 in the NC and CS groups were detected by RT-qPCR. The results showed that compared with the NC group, the gene expression of bone markers COL1A1, RUNX2, OSX, OPN and OCN were all decreased in the CS group, and the gene expression of WISP2 was also decreased. Figure 6 ).

[0154] 6. Western Blot results of osteogenic induction of BMSCs in normal individuals and CS patients

[0155] After osteogenic induction, the protein expression levels of osteogenic factors and WISP2 in the NC and CS groups were detected by Western blotting. The results showed that compared with the NC group, the protein expression of osteogenic markers RUNX2, OPN and OCN was decreased in the CS group, and the protein expression of WISP2 was also decreased. Figure 7 ).

[0156] Example 2: Investigating the Mechanism of WISP2's Influence on Osteogenic Differentiation of BMSCs

[0157] I. Experimental Materials and Methods

[0158] (a) Experimental materials

[0159] 1. Experimental equipment

[0160] Same as Example 1.

[0161] 2. Experimental reagents

[0162] Table 2-1 Main Experimental Reagents

[0163]

[0164] (II) Experimental Methods

[0165] 1. Lentiviral transfection

[0166] Preliminary experiments were conducted to determine cell density, MOI value, etc. BMSCs were seeded in 6-well plates. When the cell confluence reached 70%-80%, the cells were digested, resuspended, and re-coated. The target lentivirus volume was added to the negative control group and experimental group at an MOI of 200, and polybrene was added at 5 μg / ml. After 48 h, the original medium was discarded and fresh medium was added. After 72 h, puromycin was added at 1 μg / ml to remove untransfected cells. GFP was observed under a microscope to determine the transfection status (>80% was considered effective transfection).

[0167] 2. Osteogenic induction differentiation

[0168] Osteogenic induction was performed on transfected BMSCs, following the same steps as in Example 1.

[0169] 3. ALP staining and activity assay

[0170] 3.1 ALP staining

[0171] Same as Example 1.

[0172] 3.2 ALP Activity Assay

[0173] Same as Example 1.

[0174] 4. ARS staining and quantitative analysis of mineralized nodules

[0175] 4.1 ARS staining

[0176] Same as Example 1.

[0177] 4.2 Quantitative Analysis of Mineralized Nodules

[0178] Same as Example 1.

[0179] 5. RNA extraction, reverse transcription, and real-time quantitative PCR (RT-qPCR)

[0180] Same as Example 1.

[0181] 6. Total protein extraction and Western blot

[0182] Same as Example 1.

[0183] 7. Statistical Analysis

[0184] All data are expressed as mean ± standard deviation. Statistical analysis and graphing were performed using GraphPad Prism 10.1.2 software. Comparisons between two groups were conducted using... t The test can be either the Mann-Whitney test or a similar test. For comparisons of multiple groups, one-way ANOVA or the Kruskal-Wallis test can be used. P < 0.05 is statistically significant (*) p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001).

[0185] II. Experimental Results

[0186] 1. Knockdown of WISP2 inhibits osteogenic differentiation of BMSCs

[0187] To further investigate the regulatory role of WISP2 in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), this study employed lentivirus-mediated shRNA interference technology to silence the WISP2 gene in BMSCs. In the experimental design, three shRNA interference sequences targeting different sites specifically for the WISP2 gene were constructed. The shRNA sequence with the best gene silencing efficiency was selected by RT-qPCR. Based on this, Western blotting was used to verify protein expression levels, thereby establishing a stable WISP2-low expression cell model. Compared with the control group, all three shRNAs could knock down WISP2 gene expression, with shRNA1 showing the most significant effect. Subsequent Western blotting experiments detected its effect on WISP2 protein expression, showing that compared with the control group, shRNA1 significantly inhibited WISP2 protein expression. Figure 8 Subsequently, osteogenic differentiation was induced in BMSCs after transfection with shRNA1. ALP staining and activity assays, ARS staining and calcium nodule quantification, RT-qPCR, and Western blotting were performed on both the shWISP2 group and the control group. Results showed that, compared with the control group, ALP staining was lighter in the shWISP2 group, and ALP activity was inhibited. Figure 9 ARS staining became lighter, and the number of mineralized nodules decreased. Figure 10 RT-qPCR showed that, compared with the control group, the gene expression of osteogenic factors COL1A1, RUNX2, OSX, OPN, and OCN was suppressed. Figure 11 Western blot analysis showed that when WISP2 was knocked down, the protein expression of RUNX2, OPN, and OCN was also reduced. Figure 12 The above results indicate that knocking down WISP2 weakens the osteogenic differentiation capacity of BMSCs.

[0188] Table 2-2 shRNA sequences

[0189]

[0190] 3. WISP2 overexpression promotes osteogenic differentiation of CS-BMSCs

[0191] To elucidate the biological function of WISP2 in osteogenic differentiation of bone mesenchymal stem cells (BMSCs) from CS patients, a WISP2 overexpression vector was constructed using a lentiviral vector system. After stable overexpression of WISP2 in CS patient BMSCs via transfection, osteogenic induction was induced in both the lenti-WISP2 and CS groups, followed by ALP staining and activity assays, ARS staining and quantification of calcium nodules, RT-qPCR, and Western blotting. Results showed that compared to the CS group, the lenti-WISP2 group exhibited deeper ALP staining and promoted ALP activity. Figure 13ARS staining becomes darker, and the number of mineralized nodules increases. Figure 14 RT-qPCR showed that, compared with the CS group, the gene expression of osteogenic factors COL1A1, RUNX2, OSX, OPN, and OCN was promoted. Figure 15 Western blot analysis showed that when WISP2 was overexpressed, the protein expression of RUNX2, OPN, and OCN also increased. Figure 16 These findings indicate that overexpression of WISP2 can significantly enhance the osteogenic differentiation potential of BMSCs in CS patients.

[0192] Example 3: Study on the effect of astragaloside A-mediated WISP2 activation of the Wnt / β-catenin signaling pathway to promote osteogenic formation of BMSCs in CS patients

[0193] I. Experimental Materials

[0194] 1. Experimental apparatus

[0195] Same as Example 1.

[0196] 2. Experimental reagents

[0197] Same as Example 1.

[0198] Experimental methods

[0199] Preparation of AST solution

[0200] Weigh 20 mg of astragaloside A standard using an electronic balance and pour it into a 1.5 ml centrifuge tube. Add 500 μl of DMSO to prepare a stock solution with a concentration of 40 μg / μl. After thorough dissolution, filter through a 0.22 μm filter and store in a refrigerator protected from light.

[0201] 2. Cell proliferation experiment

[0202] CS-BMSCs were seeded at a rate of 2 × 10⁴ cells / well in a 96-well plate;

[0203] After cell adhesion, the culture medium was removed, and complete culture medium containing 0 μg / ml, 4 μg / ml, 8 μg / ml, 16 μg / ml, and 32 μg / ml AST stock solution was added respectively. The intervention was carried out for 24 h, 48 h, 72 h, and 96 h, with three replicates for each concentration and each time point.

[0204] At the time point to be tested, remove the culture medium, add 100 μl of complete culture medium containing 10% CCK8 solution, and incubate for two hours;

[0205] The OD value was measured at a wavelength of 450 nm using an ELISA reader.

[0206] 3. Effects of AST on osteogenic differentiation of CS-BMSCs

[0207] 3.1 ALP staining and ALP activity assay

[0208] (1) ALP staining: CS-BMSCs were seeded in 24-well plates. When the cell confluence reached 70-80%, the complete culture medium was removed and osteogenic induction differentiation medium containing 0 μg / ml, 4 μg / ml, 8 μg / ml, 16 μg / ml, and 32 μg / ml AST stock solution was added respectively. Each group had three replicates. The medium was changed every 3 days. Staining was performed after 14 days of induction. The specific steps were the same as in Example 1.

[0209] (2) ALP activity assay: Same as in Example 1.

[0210] 3.2 ARS staining and quantitative analysis of mineralized nodules

[0211] (1) CS-BMSCs were seeded in 24-well plates. When the cell confluence reached 70-80%, the complete culture medium was removed and osteogenic induction differentiation medium containing 0 μg / ml, 4 μg / ml, 8 μg / ml, 16 μg / ml, and 32 μg / ml AST stock solution was added respectively. Each group had three replicates. The medium was changed every 3 days. After induction for 21 days, staining was performed. The specific steps were the same as in Example 1.

[0212] (2) Quantitative analysis of mineralized nodules: Same as in Example 1

[0213] 3.3 RT-qPCR

[0214] Same as Example 1.

[0215] 3.4 Western Blot

[0216] Same as Example 1.

[0217] 4. Molecular docking of AST and WISP2

[0218] Download and save the PDB file of the 3D structure of WISP2 (AF-O76076-F1-v4) from the Alpha Fold protein structure database, and download and save the SDF file of the 3D structure of astragaloside A (Compound CID: 5488387) from Pub Chem. Upload both files to Cavity-detectionguided Blind Docking for automatic docking, and save the resulting visualization image. CB-Dock is an online molecular docking tool that can predict the binding site of a given protein and perform docking using the popular docking program Autodock Vina.

[0219] 5. A study on AST-mediated WISP2 activation of the Wnt / β-catenin pathway to promote osteogenic differentiation of BMSCs in CS patients.

[0220] To elucidate the pharmacological mechanism of astragaloside A on osteogenic differentiation of CS-BMSCs, this study focused on detecting the activation level of the Wnt / β-catenin signaling pathway and the expression characteristics of related molecules. CS-BMSCs were divided into three groups: CS group, CS+AST group, and CS+AST+DKK1 group. BMSCs were seeded in well plates. When the cell confluence reached 70-80%, osteogenic differentiation induction medium was added to the CS group, osteogenic differentiation induction medium containing 8 μg / ml AST stock solution was added to the CS+AST group, and osteogenic differentiation induction medium containing 8 μg / ml AST stock solution and 300 ng / ml DKK1 was added to the CS+AST+DKK1 group.

[0221] 5.1 ALP staining and activity assay

[0222] (1) ALP staining: Same as in Example 1.

[0223] (2) ALP activity assay: Same as in Example 1.

[0224] 5.2 ARS staining and quantitative analysis of mineralized nodules

[0225] (1) ARS staining: Same as in Example 1.

[0226] (2) Quantitative analysis of mineralized nodules: Same as in Example 1.

[0227] 5.3 RT-qPCR

[0228] Same as Example 1.

[0229] Table 3-1 RT-qPCR Primer Sequences

[0230]

[0231] The other primer sequences are the same as in Example 1.

[0232] 5.4 Western Blot

[0233] Same as Example 1.

[0234] 6. Statistical Analysis

[0235] All data are expressed as mean ± standard deviation. Statistical analysis and graphing were performed using GraphPad Prism 10.1.2 software. Comparisons between two groups were conducted using... tThe test can be either the Mann-Whitney test or a similar test. For comparisons of multiple groups, one-way ANOVA or the Kruskal-Wallis test can be used. P < 0.05 is statistically significant (*) p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001).

[0236] II. Experimental Results

[0237] 1. Cell proliferation experiment

[0238] like Figure 17 As shown, AST concentrations of 0 μg / ml, 4 μg / ml, 8 μg / ml, and 16 μg / ml all promoted the proliferation of CS-BMSCs, while 32 μg / ml showed inhibition of CS-BMSC proliferation.

[0239] 2. AST promotes osteogenic differentiation of CS-BMSCs

[0240] 2.1 Results of ALP staining and ALP activity assay

[0241] CS-BMSCs were treated with AST at concentrations of 0 μg / ml, 4 μg / ml, 8 μg / ml, 16 μg / ml, and 32 μg / ml, respectively. The ALP results after osteogenic induction showed that, compared with the control group, the staining was darker in the 4 μg / ml, 8 μg / ml, and 16 μg / ml groups, and ALP activity was increased, with the most significant increase observed at 8 μg / ml. Figure 18 ).

[0242] 2.2 ARS staining and quantitative analysis of mineralized nodules

[0243] Osteogenic differentiation of CS-BMSCs was induced by AST at concentrations of 0 μg / ml, 4 μg / ml, 8 μg / ml, 16 μg / ml, and 32 μg / ml, respectively. After 21 days of osteogenic induction, ARS staining and quantitative analysis of calcium nodules were performed. The results were consistent with ALP staining. Compared with the control group, ARS staining was darker and the number of calcium nodules increased in the 4 μg / ml, 8 μg / ml, and 16 μg / ml groups, with the most significant increase observed at 8 μg / ml. Figure 19 ).

[0244] 2.3 RT-qPCR Results

[0245] CS-BMSCs were intervened with AST at 0 μg / ml, 4 μg / ml, 8 μg / ml, 16 μg / ml, and 32 μg / ml respectively for osteogenic differentiation. After 10 days, RNA was extracted, reverse transcribed into cDNA, and RT-qPCR was performed to detect the expression levels of osteogenesis-related factors and WISP2 mRNA. The results showed that compared with the control group, the groups at 4 μg / ml, 8 μg / ml, and 16 μg / ml not only promoted the gene expression of COL1A1, RUNX2, OSX, OPN, and OCN, but also promoted the gene expression of WISP2 ( Figure 20 ).

[0246] 2.4 Western Blot results

[0247] CS-BMSCs were intervened with AST at 0 μg / ml, 4 μg / ml, 8 μg / ml, 16 μg / ml, and 32 μg / ml respectively for osteogenic differentiation. The Western Blot results showed that compared with the control group, the groups at 4 μg / ml, 8 μg / ml, and 16 μg / ml not only promoted the protein expression of RUNX2, OPN, and OCN, but also promoted the protein expression of WISP2 ( Figure 21 ).

[0248] 3. Molecular docking of AST and WISP2

[0249] As Figure 22 shown, the lower the Vina score of a general compound and the target protein, the closer the binding of the two molecules. Affinity > -4 kcal·mol-1, the binding force is extremely weak or considered non-binding; -7 kcal·mol-1 < Affinity <= -4 kcal·mol-1, the binding force is medium; Affinity <= -7 kcal·mol-1, the binding force is strong

[49] .

[0250] 4. AST mediates WISP2 to activate the Wnt / β-catenin pathway to promote osteogenic differentiation of CS-BMSCs

[0251] In this application, CS-BMSCs were divided into three groups: CS, CS+AST, and CS+AST+DKK1. DKK1 is a specific inhibitor of the Wnt / β-catenin pathway. After 14 days of osteogenic induction of the three groups of BMSCs, ALP staining and ALP activity measurement were performed. The results showed that compared with the CS group, the ALP staining in the AST group was significantly deeper and the ALP activity measurement increased. Compared with the AST group, the ALP staining in the AST+DKK1 group was significantly lighter and the ALP activity measurement was significantly reduced ( Figure 23); After 21 days of osteogenic induction, ARS staining and quantitative analysis of calcium nodules were performed on the three groups of BMSCs. The results were consistent with ALP staining. Compared with the CS group, the AST group had significantly darker ARS staining and an increased number of mineralized nodules. Compared with the AST group, the AST+DKK1 group had significantly lighter ARS staining and a significantly reduced number of mineralized nodules. Figure 24 RNA was extracted from three groups of BMSCs 10 days after osteogenic induction. RT-qPCR was used to detect the mRNA expression levels of β-catenin, COL1A1, RUNX2, OSX, OPN, OCN, and WISP2. The results showed that the gene expression of β-catenin, COL1A1, RUNX2, OSX, OPN, OCN, and WISP2 was significantly increased in the AST group. Compared with the AST group, the gene expression of β-catenin, COL1A1, RUNX2, OSX, OPN, OCN, and WISP2 was significantly inhibited in the AST+DKK1 group. Figure 25 Proteins were extracted from three groups of BMSCs 10 days after osteogenic induction. Western blot was used to detect the protein expression levels of β-catenin, RUNX2, OPN, and WISP2. The results showed that compared with the CS group, the AST group significantly promoted the expression of β-catenin, RUNX2, OPN, and WISP2 proteins. Compared with the AST group, the AST+DKK1 group showed significantly inhibited the expression of β-catenin, RUNX2, OPN, and WISP2 proteins. Figure 26 The above results indicate that AST can enhance the osteogenic differentiation capacity of CS-BMSCs by activating the Wnt / β-catenin pathway, and this capacity is likely generated through binding to WISP2.

[0252] Example 4: Astragaloside A bone-targeting injection nanosphere suspension

[0253]

[0254] The preparation method adopts the emulsification-solvent evaporation method.

[0255] 1) AST and PLGA were dissolved in 2 mL of dichloromethane; n-HAp was pre-dispersed in 0.5 mL of 0.5% polysorbate-80 solution.

[0256] Emulsify with an ultrasonic probe for 2 min (300 W, ice bath). Pour the emulsion into 50 mL of an aqueous phase containing 1% polysorbate-80 and stir at high speed for 4 h to evaporate the organic solvent.

[0257] 2) Purification and concentration: Centrifuge at 10,000 g for 10 min, discard the supernatant, and resuspend in isotonic mannitol solution. After aseptic filtration (0.22 µm), dispense into 5 mL sterile glass bottles and store at 2–8 ℃ protected from light.

[0258] Quality indicators: average particle size 240 ± 20 nm, PDI ≤0.15; encapsulation efficiency 82 ± 3%; in vitro sustained release 80% ± 5% after 72 h.

[0259] Dosage and administration: Inject 0.1 mL / kg at a single point in the local bone defect area. -1 (AST ≈ 0.5 mg·kg) -1 (1) Once every 7 days, for 4 weeks as one course of treatment.

[0260] Example 5: Astragaloside A-β-TCP 3D Printed Bone Filler Block

[0261]

[0262] The preparation method is as follows:

[0263] 1) Premixing: AST and β-TCP are mixed evenly, and gelatin-MA solution is added to make an extrudable slurry.

[0264] 2) 3D printing: Set the aperture to 400 µm, layer height to 300 µm, and print the support frame at 30 ℃.

[0265] 3) Photocrosslinking: Irradiate at 365 nm for 120 s to harden and form.

[0266] 4) Drying and γ-irradiation sterilization (25 kGy).

[0267] AST release curve: 65% cumulative release in 14 days; the scaffold completely degrades and is replaced by new bone within 8 weeks.

[0268] Example 6: Oral sustained-release capsules

[0269]

[0270] The preparation method is as follows:

[0271] 1) Mix AST, HPMC, cross-linked CMC-Na, and microcrystalline cellulose.

[0272] 2) Dry granulation (rotor granulation, 80 mesh sieve), mix with magnesium stearate and fill into empty capsules.

[0273] Dissolution behavior: Stable release of ≥ 80% in pH 6.8 buffer solution over 12 h.

[0274] Clinically recommended dosage: 1 tablet once daily for children weighing 30–60 kg; 2 tablets once daily for children weighing over 60 kg, for a course of 12 weeks.

[0275] The foregoing description of embodiments of the present invention, through which those skilled in the art are able to implement or use the present invention, will be readily apparent to those skilled in the art. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novelty disclosed herein.

Claims

1. The use of astragaloside A in the preparation of drugs for treating congenital scoliosis, characterized in that: The drug is used to promote osteogenic differentiation of bone mesenchymal stem cells (BMSCs) in patients with congenital scoliosis by upregulating the expression of WNT-1-induced signaling pathway protein 2 and activating the Wnt / β-catenin signaling pathway; and to increase alkaline phosphatase activity, mineralized nodule formation, and expression levels of osteogenic-related genes RUNX2, COL1A1, OSX, OPN, and OCN in BMSCs of patients with congenital scoliosis.

2. The use according to claim 1, characterized in that, The drug is a solution, lyophilized powder, or polylactic acid-hydroxyapatite nanosphere sustained-release formulation containing 1.0 mg / mL to 10 mg / mL astragaloside A.

3. The use according to claim 1, characterized in that, The drug further comprises a pharmaceutically acceptable carrier and, by w / w, 1% to 10% of a stabilizer, oligosaccharide, or surfactant.

4. The use according to claim 1, characterized in that, The drug is administered subcutaneously or locally intraosseously, with a single dose of 0.05 mg / kg to 2 mg / kg body weight, every 5 to 10 days, for 4 to 12 weeks.

5. The use according to claim 1, characterized in that, The drug is indicated for the treatment or prevention of osteoporosis or insufficient bone mineralization in patients aged 4–25 years with congenital scoliosis, a Cobb angle ≥10°, and a DXA T value ≤-2.0 SD.

6. The use according to claim 1, characterized in that, The drug was used to expose BMSCs from patients with congenital scoliosis to a final concentration of 4 µg / mL to 16 µg / mL in vitro for 7 to 21 days, thereby increasing the expression of WISP2 and β-catenin mRNA by at least 1.5 times.

7. The use according to claim 1, characterized in that, The drug is used in combination with 10–100 ng / mL fibroblast growth factor-2 or bone morphogenetic protein-2 to synergistically enhance osteogenic differentiation of CS-BMSCs.

8. The use according to claim 1, characterized in that, The drug is a pH-sensitive or esterase-degradable microsphere formulation with a particle size of 100 nm to 500 nm, and it releases more than 80% of astragaloside A within 72 h.

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