Application of small molecule ISX-9 in promoting mesenchymal stem cell osteogenic differentiation and bone injury regeneration and repair

Abstract

The invention provides application of isoxazole 9 (ISX-9) in promoting mesenchymal stem cell osteogenic differentiation and bone injury regeneration and repair. ISX-9 is a small molecular compound and has the characteristics of easiness in obtaining, controllable structure, low cost and the like. In-vitro experiments show that ISX-9 has the capabilities of inducing mesenchymal stem cell osteogenic differentiation and promoting mineralized nodule formation; in-vivo experiments show that the hydrogel wrapping ISX-9 can promote mouse skull defect regeneration and repair, which indicates that ISX-9 has good osteogenic inductivity and is a potential drug for promoting bone regeneration and repair. Therefore, ISX-9 can promote mesenchymal stem cell osteogenic differentiation and is applied to bone tissue regeneration and repair. The invention also provides a direct action target and an action mechanism of the ISX-9 for promoting osteogenic differentiation. The invention further provides a novel ISX-9 drug sustained-release delivery system, and theoretical research and clinical application of the ISX-9 drug sustained-release delivery system are facilitated.

Description

Technical Field

[0001] The present invention relates to the field of stem cell technology, and more specifically, to the application of ISX-9 in promoting the osteogenic differentiation of mesenchymal stem cells and the regeneration and repair of bone damage. Background Art

[0002] In recent years, mesenchymal stem cells (MSCs) have garnered increasing attention as a model for studying bone tissue regeneration and repair. Stem cells are multipotent cells that possess the ability to self-renew in vivo and, under appropriate conditions, differentiate into a variety of cell types, including osteoblasts. Bone marrow-derived mesenchymal stem cells (BMSCs) are the most extensively studied cell type in bone tissue engineering. However, obtaining and culturing BMSCs in vitro presents significant challenges in clinical application. Adipose-derived mesenchymal stem cells (ADSCs), like BMSCs, originate from the mesoderm and possess the same robust osteogenic differentiation potential. ADSCs also offer the advantages of a wide range of sources, ease of harvest, and minimal damage to the donor site. ADSCs are a promising candidate for seed cells in future bone tissue engineering, holding broad scientific significance and application prospects. Therefore, ADSCs were selected as the cell model for this study. This method is applicable to all mesoderm-derived mesenchymal stem cells, as well as induced pluripotent stem cells (iPS cells).

[0003] Small molecule compounds have become a research frontier in the field of bone regeneration due to their unique advantages such as targeted regulation of stem cell fate, controllable costs, and easy scalable preparation. Developing an efficient, safe, and economical small molecule bone repair drug is an important approach to solving the clinical dilemma of bone regeneration. ISX-9 is composed of isoxazole and benzene rings, with a molecular weight of 234.27 Da. Due to its small molecular weight and high lipid solubility, it can quickly penetrate the cell membrane to exert its effect. Recent studies have shown that ISX-9 exhibits significant biological activity in various cell types, especially in the regulation of stem cell fate, cell survival, and proliferation. ISX-9 not only promotes calcium influx by activating voltage-gated calcium channels and N-methyl-D-aspartate (NMDA) receptors, inducing neural stem cell differentiation into neurons, but also crosses the blood-brain barrier and, depending on the activation of myocyte enhancer 2 (Mef2), promotes neural stem cell proliferation and neurogenesis in the hippocampus (Luis EB Bettio, et al. ISX-9 can potentiate cell proliferation and neuronal commitment in therat dentate gyrus [J]. Neuroscience, 2016, 332:212-222.). Furthermore, ISX-9 plays a key role in regulating the fate determination of mesodermal-derived cells. ISX-9 activates both canonical and non-canonical WNT signaling pathways at different stages of cardiac progenitor cell (CPC) differentiation, promoting cardiac lineage differentiation. ISX-9 can also determine the differentiation direction of enteroendocrine cells (EECs) and the fate of intestinal cell lineages through the regulation of calcium signaling (Tsakmaki A, et al. ISX-9 manipulates endocrine progenitor fate revealing conserved intestinal lineages in mouse and human organoids[J]. Molecular Metabolism, 2020, 34.). Although ISX-9 plays an important role in regulating neurogenesis and neuronal function, its role in the osteogenic differentiation of mesenchymal stem cells and the regeneration and repair of bone damage has not been reported. Summary of the Invention

[0004] The present invention aims to overcome the aforementioned deficiencies and shortcomings of the prior art and to provide the use of ISX-9 in promoting the osteogenic differentiation of mesenchymal stem cells and the regeneration and repair of bone damage. Research in this invention demonstrates that ISX-9 has the ability to induce the osteogenic differentiation of mesenchymal stem cells and promote the formation of mineralized nodules. In vivo experiments have shown that hydrogels encapsulating ISX-9 can promote the regeneration and repair of skull defects in mice, demonstrating that ISX-9 has excellent osteogenic induction and is a potential drug for promoting bone regeneration and repair. Furthermore, we have discovered the mechanism and target of ISX-9's osteogenic differentiation promotion and have developed an ISX-9 sustained-release system. This overcomes the technical bottlenecks of existing hydrophobic small molecule drugs, such as low local delivery efficiency and short duration of action, and provides a new target and intelligent drug delivery strategy for the clinical treatment of bone damage.

[0005] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0006] This study used the small molecule drug ISX-9 to investigate the effects of varying concentrations on the osteogenic differentiation and mineralization of ADSCs. Furthermore, ISX-9 was encapsulated in a methacrylate glycol chitosan (MeGC) hydrogel developed by our research team and implanted in a non-self-healing mouse skull defect model to test ISX-9's ability to promote bone regeneration in vivo, providing a foundation for promoting ISX-9's clinical application. Furthermore, through thermostable proteomics combined with mass spectrometry and transcriptome sequencing, we identified the direct downstream targets and mechanisms of action of ISX-9. Based on the application characteristics of ISX-9, we designed a sustained-release system using poly(lactic-co-glycolic acid) (PLGA), an FDA-approved excipient, to promote its application and translation.

[0007] In vitro studies have demonstrated that ISX-9 exhibits excellent biocompatibility within a concentration range of 5-30 μM and can upregulate the gene and protein expression of osteogenic markers in ADSCs, such as alkaline phosphatidylcholine (ALP), RUNX2, osteopontin (OPN), and osteocalcin (OCN). Alizarin red staining and quantitative analysis indicate that ISX-9 can induce the formation of mineralized nodules in ADSCs. The optimal osteogenic induction concentration is 20 μM, at which ISX-9's ability to promote osteogenic differentiation is superior to that of bone morphogenetic protein 2 (BMP2), a currently clinically approved drug for the treatment of bone defects.

[0008] In vivo experimental studies have shown that hydrogels encapsulating ISX-9 can promote the regeneration and repair of skull defects in mice. μCT three-dimensional reconstruction images 6 weeks after surgery showed that ISX-9 in the concentration range of 50-400 μM can promote the repair of skull defects in mice, among which the 100 and 200 μM groups showed better effects in promoting bone regeneration and repair, indicating that ISX-9 has good osteogenic induction and is a potential drug for promoting bone regeneration and repair.

[0009] Therefore, the present invention provides the use of ISX-9 in promoting osteogenic differentiation of mesenchymal stem cells. The chemical structure of ISX-9 is shown below: .

[0010] Furthermore, the in vitro working concentration of ISX-9 is 5 to 30 μM.

[0011] Preferably, the working concentration of ISX-9 in promoting osteogenic differentiation in vitro is 15-25 μM.

[0012] Most preferably, 20 μM ISX-9 has the strongest effect on promoting osteogenic differentiation of ADSCs.

[0013] The present invention also provides the use of a hydrogel encapsulating ISX-9 in promoting osteogenic differentiation of mesenchymal stem cells. The chemical structure of the ISX-9 is shown below: .

[0014] Furthermore, the hydrogel is selected from one or more of methacrylate glycol chitosan (MeGC) hydrogel, gelatin hydrogel or hyaluronic acid hydrogel.

[0015] Furthermore, the in vitro working concentration of the MeGC hydrogel-encapsulated ISX-9 is 0-600 μM.

[0016] Preferably, the working concentration of the MeGC hydrogel-encapsulated ISX-9 in promoting osteogenic differentiation is 50-400 μM.

[0017] Most preferably, the ISX-9 concentration in the MeGC hydrogel is 200 μM, which has the strongest effect on promoting osteogenic differentiation.

[0018] Furthermore, the mesenchymal stem cells are selected from one or more of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells or induced pluripotent stem cells (iPSCs).

[0019] The present invention also provides the use of ISX-9 in preparing a bone injury regeneration and repair product, wherein the bone injury is one or more of bone defect, bone nonunion, or delayed bone healing, and the chemical structure of the ISX-9 is as follows: .

[0020] The present invention also provides the use of a hydrogel encapsulating ISX-9 in preparing a bone injury regeneration and repair product, wherein the chemical structure of the ISX-9 is as follows: .

[0021] Furthermore, the hydrogel is selected from one or more of methacrylate glycol chitosan (MeGC) hydrogel, gelatin hydrogel or hyaluronic acid hydrogel.

[0022] Furthermore, the working concentration of the MeGC hydrogel-encapsulated ISX-9 in promoting the regeneration and repair of mouse skull defects is 50-400 μM.

[0023] Preferably, when the concentration of ISX-9 in the hydrogel is 100 and 200 μM, ISX-9 has the strongest ability to promote the regeneration and repair of mouse skull defects.

[0024] The present invention also provides the use of an ISX-9 sustained-release drug delivery system in the preparation of a bone injury regeneration and repair product. The chemical structure of the ISX-9 is shown below: .

[0025] Furthermore, the drug sustained-release delivery system includes one or more of microspheres, micelles or liposomes.

[0026] Furthermore, the application of ISX-9 binding to breakpoint cluster region (BCR) protein in promoting osteogenic differentiation of mesenchymal stem cells is characterized in that the ISX-9 directly targets BCR protein to promote osteogenic differentiation of mesenchymal stem cells through the RAP1 signaling pathway.

[0027] The present invention also provides a method for inducing osteogenic differentiation of mesenchymal stem cells, characterized by using an osteogenic differentiation medium containing ISX-9 to induce osteogenic differentiation of the mesenchymal stem cells. The osteogenic differentiation medium is a conventional medium in the art (primarily containing β-glycerophosphate and ascorbic acid), and ISX-9 is added as an osteogenic differentiation promoter. The ISX-9 concentration is 15 to 25 μM, and the induction time is 7 to 14 days. The chemical structure of ISX-9 is shown below: .

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] This invention provides the use of isoxazole 9 (ISX-9) in promoting the osteogenic differentiation of mesenchymal stem cells and the regeneration and repair of bone damage. ISX-9 is a small molecule compound with the advantages of easy availability, controllable structure, and low cost. In vitro experiments demonstrate that ISX-9 can induce the osteogenic differentiation of mesenchymal stem cells and promote the formation of mineralized nodules. In vivo experiments show that a hydrogel encapsulating ISX-9 can promote the regeneration and repair of skull defects in mice, demonstrating that ISX-9 has excellent osteogenic induction and is a potential drug for promoting bone regeneration and repair. Therefore, ISX-9 can promote the osteogenic differentiation of mesenchymal stem cells and be used in bone tissue regeneration and repair. This invention also provides the direct target and mechanism of action of ISX-9 in promoting osteogenic differentiation. Furthermore, this invention provides a novel sustained-release drug delivery system for ISX-9, which will facilitate the transition of this invention from theoretical research to clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The results of the CCK8 cytotoxicity experiment on ISX-9 are shown.

[0031] Figure 2 Figure 3 ALP staining and quantification after ISX-9 and hADSCs were co-cultured for 3 days.

[0032] Figure 3 Figure 3 ALP staining and quantification after ISX-9 and hADSCs were co-cultured for 7 days.

[0033] Figure 4 Alizarin Red S staining and quantification of mineralized nodules after ISX-9 and hADSCs were co-cultured for 14 days.

[0034] Figure 5 This is the result of the effect of ISX-9 on the relative expression of osteogenic differentiation genes in hADSCs.

[0035] Figure 6 This is the result of the effect of ISX-9 on the protein level of hADSCs osteogenic differentiation.

[0036] Figure 7 The results of CCK8 cytotoxicity experiments in three-dimensional culture of MeGC hydrogels encapsulating ISX-9.

[0037] Figure 8 ALP staining and quantification of ISX-9-encapsulated MeGC hydrogel 3D culture.

[0038] Figure 9 Alizarin Red S staining and quantification of mineralized nodules in three-dimensional culture of ISX-9-encapsulated MeGC hydrogels.

[0039] Figure 10 The results of bioinformatics analysis and ISX-9 target verification are shown.

[0040] Figure 11 MeGC hydrogel encapsulating ISX-9 promotes regeneration and repair of skull defects in mice.

[0041] Figure 12 Characterization of PLGA encapsulated ISX-9-MeGC sustained-release system. DETAILED DESCRIPTION

[0042] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0043] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0044] ISX-9, CAS No: 832115-62-5, was purchased from AbMole, USA. The main research routes of the present invention are as follows:

[0045] Research route 1: The role of ISX-9 in the osteogenic differentiation of ADSCs.

[0046] Specific experimental plan:

[0047] (1) Human ADSCs (hADSCs) were purchased from the National Stem Cell Transformation Resource Bank (ZB10DGAC) and cultured in Dulbecco's Minimum Essential Medium (DMEM) containing 10% fetal bovine serum (FBS), 1% penicillin / streptomycin (P / S), 2 mM glutamine, 10 ng / mL fibroblast growth factor (FGF), and 1% non-essential amino acids (NEAA). hADSCs were cultured and divided into two groups. When the cell growth area reached 60% of the bottom area of the culture dish, one group was placed in ordinary culture medium, and the other group was placed in complete culture medium containing different concentrations (5, 10, 15, 20, 25, 30, and 50 μM) of ISX-9. The effects on hADSC cell viability were detected by CCK-8 assay after 1, 3, and 7 days of culture.

[0048] (2) hADSCs were cultured in osteogenic induction medium with or without different concentrations of ISX-9, and the expression of BCR and osteogenic differentiation-related markers was detected by RT-qPCR and Western blot.

[0049] (3) Collect cells at different times or perform alkaline phosphatase staining or alizarin red staining on the cells, and use relevant methods and techniques to evaluate the osteogenic differentiation of the cells.

[0050] Research Route 2: In vivo experiments explore the role of ISX-9 in bone regeneration and repair.

[0051] The experimental plan involved encapsulating ISX-9 or ISX-9@PLGA nanospheres at the optimal concentrations tested in vitro in MeGC hydrogel and implanting them into skull defects in mice. Six weeks after surgery, the mice were sacrificed, and skull tissue was extracted. Bone defect healing was monitored using in vitro microCT. Trabecular bone volume and bone density were analyzed using CTan image processing software. After decalcification, the bone tissue was paraffin-embedded and sectioned (5 µm) for histological evaluation, including H&E staining, Masson's trichrome staining, and immunohistochemistry. The biomechanical properties of the newly formed bone were assessed using a three-point bend test.

[0052] Example 1 Study on the effect of ISX-9 on osteogenic differentiation and mineralization of ADSCs in vitro

[0053] 1. Experimental Methods

[0054] 1. Obtaining hADSCs and preparing osteogenic induced medium (OM)

[0055] The hADSCs used in the present invention were purchased from the National Stem Cell Transformation Resource Bank (ZB10DGAC).

[0056] Osteogenic medium (OM): Add 10% FBS and 1% double-antibody to L-DMEM, then add 10 mM sodium β-glycerophosphate and 50 μg / mL vitamin C. Store sodium β-glycerophosphate at -20°C. Prepare the vitamin C stock solution for immediate use.

[0057] 2. Preparation of ISX-9

[0058] ISX-9 was purchased from AbMole, USA. According to the product instructions, 10 mg of ISX-9 was dissolved in 426.86 μL of dimethyl sulfoxide (DMSO) to prepare a 100 mM (23.43 mg / mL) ISX-9 stock solution, which was then aliquoted and stored at -80°C. ISX-9 was serially diluted to the corresponding working concentrations in L-DMEM medium supplemented with 10% fetal bovine serum, 1% penicillin / streptomycin, 2 mM glutamine, 10 ng / mL fibroblast growth factor (FGF), and 1% non-essential amino acids (NEAA) for subsequent in vitro experiments. Preparation was immediate.

[0059] 3. Cytotoxicity assay

[0060] hADSCs were cultured and digested with 0.25% trypsin and diluted with complete medium to a cell density of 2×10 4A cell suspension (100 μL / well) was seeded into each well of a 96-well plate and incubated at 37°C, 5% CO₂, in L-DMEM complete medium. After 1 day of culture, the medium was replaced with L-DMEM complete medium containing 0, 5, 10, 15, 20, 25, 30, or 50 μM ISX-9. Six replicate wells were set up for each experimental group. The medium was changed every 2 days. The cells were assayed on days 1, 3, and 7. At the corresponding time points, the cells were removed, the culture medium discarded, and washed twice with sterile PBS. The effect of different concentrations of ISX-9 on the proliferation of hADSCs was determined by CCK8 assay. 90 μL of fresh culture medium and 10 μL of CCK8 reagent were added to each experimental well and blank control well, followed by incubation in the dark for 1 hour. The absorbance of the replicate wells in each experimental group at 450 nm was measured in triplicate using an automated multifunctional enzyme-linked immunosorbent assay (ELISA). The average value was calculated, recorded, analyzed, and compared.

[0061] 4. Alizarin Red S staining and quantification

[0062] Alizarin red S was used to qualitatively analyze the mineralized nodule staining results, and acetic acid extraction was used to semi-quantitatively analyze the ability of ISX-9 to induce hADSCs to form mineralized nodules.

[0063] (1) Alizarin Red S staining

[0064] hADSCs were routinely digested with 0.25% trypsin, counted, and resuspended at 3×10 4 Cells were seeded at a density of 100 μg / well in a 24-well plate, with four replicates per group. When the cell growth density reached 50%-60%, the cell culture medium was replaced with osteogenic induction medium (complete medium supplemented with 10 mM sodium β-glycerophosphate and 50 μg / mL vitamin C) containing 0, 10, 15, 20, 25, 30, and 50 μM ISX-9 and BMP2 (200 ng / ml). The medium was changed every 3 days. On the 14th day, the medium was discarded and the cells were washed three times with PBS buffer. 300 μL of 4% paraformaldehyde fixative was added to each well and fixed for 20 min. After fixation, each experimental well in each group was washed three times with PBS buffer. 300 μL of 1% (volume fraction) Alizarin Red S solution (pH 4.2) was added according to the product instructions. The cells were allowed to stand at room temperature in the dark for 10 min, after which the stain was aspirated. The cells were washed repeatedly with PBS buffer until the supernatant was colorless or no longer changed in color. The cells were then air-dried at room temperature and observed under a scanning microscope and stereomicroscope.

[0065] (2) Semi-quantitative analysis of mineralized nodules

[0066] Add 800 μL of 10% (v / v) acetic acid to each well and shake on a shaker for 30 minutes. Add the solution to a 1.5 mL EP tube and vortex for 30 seconds. Add 500 μL of mineral oil to the top layer, heat at 85°C for 10 minutes, and place on ice for 5 minutes. Centrifuge at 20,000 × g for 15 minutes, aspirate 500 μL of the middle layer, add 200 μL of 10% (v / v) ammonia water, and mix thoroughly. Transfer 150 μL to a 96-well plate and measure absorbance at 405 nm using a microplate reader.

[0067] 5. Cell ALP activity detection

[0068] 2×10 4 hADSCs were plated and incubated in a cell culture incubator at 37°C, 5% CO2 for 24 hours to allow cell attachment. After cell attachment, osteogenic induction medium containing varying concentrations of ISX-9 (0, 10, 15, 20, 25, 30, and 50 μM) and BMP2 (200 ng / ml) was added. Four replicate wells were set up in each group, and the medium was changed every three days. ALP staining and quantification were performed on days 3 and 7 of culture.

[0069] (1) Alkaline phosphatase staining

[0070] After removing the culture medium from the 24-well plate, wash it three times with PBS and add 300 μL of 4% paraformaldehyde solution to fix the cells for 30 minutes. Prepare the ALP staining working solution according to the instructions of the ALP color development kit. After fixation, wash the plate twice with PBS and add 300 μL of ALP staining working solution to each well. Stain at room temperature in the dark for 10-30 minutes. Terminate the color development reaction with ddH2O. Scan with a scanner and observe under a stereomicroscope.

[0071] (2) Quantitative detection of ALP activity

[0072] hADSCs were collected at 2×10 4 The cells were seeded at a density of 100 μL / well in a 24-well plate and cultured to a cell density of 70%-80%. The culture medium was then discarded and replaced with osteogenic induction medium containing ISX-9. The medium was changed every 3 days. On the 3rd and 7th days of culture, the culture medium was discarded, the cells were rinsed three times with PBS buffer, 100 μL of protein lysis working solution was added, and the cells were lysed on ice for 0.5 h. The lysed cells were collected into a sterile EP tube and centrifuged at 13,000 rpm and 4°C for 10 min. 80 μL of the supernatant was collected and the total protein amount and alkaline phosphatase content were detected using a total protein quantification kit and an alkaline phosphatase detection kit, respectively. The ratio of ALP concentration to total protein concentration was quantitatively calculated according to the product instructions to obtain the ALP activity of each group (unit: U / g protein).

[0073] 2. Results

[0074] 1. ISX-9 cytotoxicity test results

[0075] The results of CCK-8 cytotoxicity assay showed that ( Figure 1 Compared with the control group, at 1 day, the culture medium containing ISX-9 had no effect on the proliferation of hADSCs (P>0.05); at 3 days, the culture medium containing 5 μM ISX-9 promoted the proliferation of hADSCs (P<0.05), and there was no statistical difference in cell viability between the 10, 15, 20, 25, 30, and 50 μM ISX-9 groups and the control group (P>0.05); at 7 days, the OD values of the 5, 10, 15, and 20 μM ISX-9 groups were higher than those of the control group, but the differences were not statistically significant (P>0.05), the OD values of the 25 and 30 μM ISX-9 groups were lower than those of the control group, and the cell viability was low, but the differences were not statistically significant (P>0.05), and the OD value of the 50 μM ISX-9 group was lower than that of the control group, and the difference was statistically significant (P<0.05). μM) had no cytotoxicity to hADSCs, and even at 3 days, low concentrations of ISX-9 could enhance the proliferation ability of hADSCs.

[0076] 2. Effect of ISX-9 on ALP activity of hADSCs

[0077] Figure 2 、 3 The following are the results of ALP staining and quantification on days 3 and 7 of osteogenic induction. The results show that on day 3, ALP staining was deeper in the positive control group (BMP2) than in the control group. ALP staining in groups treated with different concentrations of ISX-9 was deeper than in the BMP2 group, with the 20 μM ISX-9 group showing the deepest ALP staining. The trend of ALP activity quantitative analysis was consistent with the staining results. ALP activity in the BMP2 group was approximately 2-fold higher than in the control group. The 10 and 50 μM ISX-9 treatment groups were significantly higher than in the control group, but were essentially the same as in the BMP2 group, with no statistical significance (P>0.05). However, the 15, 20, 25, and 30 μM ISX-9 treatment groups showed significant increases compared to the BMP2 group (P<0.05). The trends in ALP staining and quantification results on day 7 were similar to those on day 3. ALP expression in the ISX-9 treatment groups at 15, 20, 25, and 30 μM was significantly higher than that in the control group and the positive control group, the BMP2 group (P < 0.05). The qualitative staining and activity quantitative assays for ALP on days 3 and 7 indicate that ISX-9 at different concentrations can promote ALP expression in hADSCs, with 15-25 μM being the optimal concentration range and 20 μM being the optimal concentration.

[0078] 3. Mineralized nodule staining and semi-quantitative analysis results

[0079] hADSCs were co-cultured with ISX-9 for 14 days and then stained with Alizarin Red S. Figure 4 As shown in the figure, the ISX-9 group at a safe drug concentration was able to form more and larger mineralized nodules than the control group. The semi-quantitative analysis results of Alizarin Red ( Figure 4 ) showed that the 20 μM ISX-9 group achieved the highest mineralization, while 15, 20, 25, and 30 μM ISX-9 promoted significantly higher levels of hADSCs than the control and BMP2 groups (P < 0.05). The 10 and 50 μM ISX-9 groups showed no statistically significant difference in mineralization compared to the BMP2 group (P > 0.05), but were significantly higher than the control group. Alizarin Red S qualitative staining and semiquantitative analysis of mineralized nodules revealed that 15-25 μM ISX-9 was the most effective in inducing hADSC mineralized nodule formation, with 20 μM being the optimal osteogenic concentration.

[0080] Example 2 Effects of ISX-9 on the relative expression and protein levels of hADSCs osteogenic differentiation genes

[0081] 1. Experimental Methods

[0082] 1. Reverse Transcription-qPCR method was used to detect the relative expression levels of related genes during the osteogenic differentiation of hADSCs.

[0083] (1) Extraction of total RNA using Trizol

[0084] After discarding the old medium from the 24-well plate and washing with PBS, add 500 μL of Trizol lysis buffer to each well. Dissolve the cells thoroughly by pipetting, and transfer the solution to a 1.5 mL RNase-free EP tube. Add 200 μL of chloroform to each tube, mix thoroughly by inversion, and let it stand at room temperature for 3 minutes. Centrifuge at 12,000 × g, 4°C for 15 minutes, and carefully transfer the supernatant to a 1.5 mL enzyme-free centrifuge tube. Add an equal volume of absolute ethanol to the tube, mix thoroughly by pipetting, and transfer the sample to an RNA spin column. Centrifuge at 4,000 × g for 1 minute and discard the waste solution. Add 500 μL of Wash Buffer 1 to the spin column, centrifuge at 12,000 × g, 4°C for 1 minute, and discard the waste solution. Add 500 μL of Wash Buffer 2 to the spin column, centrifuge at 12,000 × g, 4°C for 1 minute, and discard the waste solution. Return the spin column to the collection tube and centrifuge at 12,000 × g at 4°C for 1 minute to remove any residual waste liquid. Transfer the spin column to a new 1.5 mL RNase-free centrifuge tube and air-dry it with the lid uncovered for 2 minutes. Add 20 μL of RNase-free water to the spin column, incubate at room temperature for 2 minutes, and then centrifuge at 12,000 × g at 4°C for 1 minute to obtain total RNA.

[0085] RNA concentration was determined using a NanaDrop spectrophotometer. Three measurements were taken of 1 μL of the extracted total RNA sample, averaging the concentration. The ratio of the OD values at 260 nm and 280 nm, as measured by the instrument, indicated the RNA purity. A ratio between 1.8 and 2.0 indicated a high level of total RNA purity, meeting the experimental requirements for RT-qPCR.

[0086] A 20 μL reaction system was prepared according to the TAKARA kit instructions, ensuring consistent RNA concentration across samples. Reverse transcription was then performed on a standard PCR instrument (37°C for 15 min, 85°C for 5 s) to synthesize cDNA.

[0087] (2) Fluorescence quantitative PCR (qPCR)

[0088] 1) Primer design

[0089] The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. The sequences of all primers involved in the Real-Time PCR in this experiment are shown in Table 2.

[0090] 2) System

[0091] According to the instructions of the Novozymes kit, 20 μL reaction system was prepared as shown in the table below for qPCR detection.

[0092] Table 1 qPCR reaction system

[0093]

[0094] 3) qPCR reaction conditions: PCR reactions were performed in a real-time fluorescence quantitative PCR instrument using the following program: pre-denaturation (95°C, 30 s); denaturation (95°C, 10 s); annealing and extension (60°C, 30 s) for 40 cycles.

[0095] 4) Real-time PCR results analysis: Three replicate wells (technical replicates) were set up for the same sample, and the Ct values of each sample in the control group and each experimental group were measured to calculate the relative expression of the target gene ( ), the experiment was repeated 3 times (experimental replicates).

[0096] Table 2 Sequence primers used for RT-qPCR

[0097]

[0098] Note: GAPDH: glycerol-3-phosphate dehydrogenase; RUNX2: Runt-related transcription factor 2; BCR: breakpoint cluster region protein; OSX: osteoblast-specific transcription factor; OPN: osteopontin; OCN: osteocalcin.

[0099] 2. Western blot detection

[0100] 1x10 cells were seeded in a 12-well plate. 5 After 24 hours, the hADSCs were cultured in duplicate, with the culture medium removed. Four replicate wells were plated in each group and replaced with osteogenic mineralization induction medium containing varying concentrations of ISX-9 and BMP2. The medium was changed every three days. Samples were harvested on day 7, the original culture medium discarded, and the cells were washed three times with PBS. The PBS in the plate was aspirated thoroughly, and RIPA lysis buffer containing PMSF and protease phosphatase inhibitors was prepared. 100-120 μL of RIPA lysis buffer was added to each well and the cells were lysed on ice for 30 minutes. The lysed mixture was collected with a cell scraper and transferred to an EP tube. The cells were pre-cooled to 4°C and centrifuged at 13,000 rpm for 15 minutes. The pellet was discarded and the supernatant collected. The total protein concentration was determined and balanced using a BCA assay. An appropriate amount of loading buffer was added, mixed, and heated in a 100°C water bath or metal bath for 10 minutes before transfer to a -80°C freezer for storage.

[0101] Electrophoresis: Prepare SDS-PAGE gel, set the sample load per well (20 μg), set the constant voltage to 80 V for the stacking gel and 120 V for the separating gel, and start electrophoresis.

[0102] Transfer: After electrophoresis is completed, first cut an NC membrane of appropriate size, and then use a Bio-Rad transfer apparatus to transfer the proteins separated on the SDS-PAGE gel to the NC membrane using a 300 mA constant current wet transfer method. The transfer time is adjusted according to the molecular weight of the corresponding protein indicator. The larger the molecular weight, the longer the transfer time.

[0103] Membrane Blocking and Antibody Incubation: After transfer, block the NC membrane with pre-prepared 5% skim milk for 1 hour at room temperature on a shaker. Rinse with TBST for 5 minutes after blocking, discard the rinse buffer, aspirate dry, and incubate with the target protein's primary antibody, prepared according to the manufacturer's instructions, overnight at 4°C on a shaker. After primary antibody incubation, rinse the membrane three times with TBST for 10 minutes at room temperature on a fast shaker. Prepare the secondary antibody with 5% skim milk and add it to the incubation box. Incubate at room temperature on a slow shaker for 1 hour. After secondary antibody incubation, rinse the membrane three times with TBST for 10 minutes at room temperature on a fast shaker. Prepare ECL chemical development solution and incubate the NC membrane containing the target protein in the solution for 1 minute in the dark. Remove the membrane with flat-nose forceps, aspirate the excess solution, and develop the membrane using a developer. Bands are qualitatively observed and recorded. Bands on the membrane are quantitatively analyzed and compared using Image J software.

[0104] 2. Results

[0105] 1. Expression of genes related to hADSCs osteogenic differentiation

[0106] The relative expression levels of hADSCs osteogenic differentiation genes were detected by qPCR. We detected RUNX2 on the third day of osteogenic differentiation, OPN on the 7th day of osteogenic differentiation, and OCN on the 14th day of osteogenic differentiation. Figure 5As shown in the results, RUNX2 gene expression was high in the BMP2 group and the groups treated with different concentrations of ISX-9 compared with the control group (P < 0.05). RUNX2 expression in the 15, 20, 25, and 30 μM ISX-9 groups was significantly higher than that in the BMP2 group (P < 0.05). RUNX2 expression in the 10 and 50 μM ISX-9 groups was slightly lower than that in the BMP2 group (P > 0.05). Regarding OPN gene expression, compared with the control group, OPN expression was high in the BMP2 group and the groups treated with different concentrations of ISX-9, and the differences were statistically significant (P < 0.05). There was no statistical difference in the relative expression of OPN gene between the 10 and 30 μM ISX-9 groups and the control group (P > 0.05). The OPN expression in the 15, 20, and 25 μM ISX-9 groups was significantly higher than that in the BMP2 group, while the OPN expression in the 50 μM ISX-9 group was slightly lower than that in the BMP2 group, and the differences were statistically significant (P < 0.05). At 14 days, OCN expression was high in the BMP2 group and the groups treated with different concentrations of ISX-9, and the differences were statistically significant (P < 0.05). The OCN gene expression in the 15, 20, 25, and 30 μM ISX-9 groups was higher than that in the BMP2 group, and the differences were statistically significant (P < 0.05). The expression of OCN genes in the ISX-9 group was essentially the same as that in the BMP2 group, with no statistically significant difference. The qPCR results indicate that ISX-9 within a safe concentration range can promote the expression of hADSCs osteogenic differentiation-related genes, with 20 μM ISX-9 exhibiting the strongest osteogenic differentiation-promoting effect.

[0107] 2. Western blot test results

[0108] Western blot test results Figure 6 As shown in the figure, we detected the protein levels of RUNX2 and OSX, key transcription factors of osteogenic differentiation, on the 7th day of osteogenic differentiation. The results are as follows: The expression levels of RUNX2 and OSX proteins in the ISX-9 group were significantly higher than those in the control group, and the differences were statistically significant (P < 0.05). This indicates that ISX-9 can promote the expression of osteogenic-related proteins RUNX2 and OSX, thereby affecting the osteogenic differentiation of hADSCs.

[0109] Example 3 ISX-9-encapsulated MeGC hydrogel promotes osteogenic differentiation of hADSCs

[0110] To better simulate the three-dimensional cell growth environment in vivo, this study used MeGC hydrogels to encapsulate hADSCs. ISX-9 was then loaded onto the MeGC hydrogels to investigate the ability of ISX-9 to promote osteogenic differentiation of ADSCs in this three-dimensional environment. This also provides a reference for the safe and effective drug concentration ranges in in vivo models.

[0111] 1. Experimental Methods

[0112] 1. MeGC hydrogel three-dimensional cell culture and CCK8 detection

[0113] Dissolve the MeGC hydrogel freeze-dried powder in PBS at a mass volume percentage of 4%. Digest the ADSCs with trypsin and add an equal amount of complete culture medium to terminate the digestion. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 180 g for 3 min to collect the cell pellet. 6 ADSCs hydrogel suspensions were prepared using 2% MeGC hydrogel, 6 μM riboflavin, and the corresponding drug concentrations. 40 μL of the mixed hydrogel was aspirated and dropped onto a polytetrafluoroethylene plate and irradiated with a blue light curing lamp (Carent LY-A180) for 40 s. The cured hydrogel was transferred to an uncoated 48-well plate, and 800 μL of complete culture medium was added to each well. The plates were cultured in a cell incubator and the medium was changed every 2-3 days. On days 1, 3, and 7 of culture, the effects of different concentrations of ISX-9 on hADSC proliferation were assessed using a CCK-8 assay. 900 μL of fresh culture medium and 100 μL of CCK-8 reagent were added to each experimental well and a blank control well in the dark. The plates were incubated in a dark incubator for 3 h. The absorbance of the replicate wells in each experimental group at 450 nm was measured three times using an automated multifunctional enzyme-linked immunosorbent assay (ELISA) reader. The average value was calculated, recorded, analyzed, and compared.

[0114] 2. Alkaline phosphatase (ALP) staining of MeGC hydrogel 3D cells

[0115] MeGC hydrogels, which had been inducing osteogenic growth for 7 days, were washed twice with PBS and fixed with 300 µL of 4% paraformaldehyde for 20 minutes. ALP staining solution was prepared according to the instructions of the ALP colorimetric kit. 300 µL of the solution was added and the cells were incubated at 37°C in the dark for a period of time. The total reaction time depended on the reaction quality. To terminate the reaction, the ALP staining solution was completely aspirated, and the cells were washed twice with ddH2O to terminate the reaction. The staining results were observed using a stereo microscope and photographed for preservation.

[0116] 3. Alizarin red staining (ARS) of MeGC hydrogel 3D cells

[0117] MeGC hydrogels, which had been inducing osteogenic growth for 14 days, were washed twice with PBS and fixed with 300 µL of 4% paraformaldehyde for 20 minutes. 300 µL of Alizarin Red stain was added and allowed to react at room temperature for 10 seconds. The hydrogels were then washed with PBS on a shaker for 24 hours to remove any excess stain, changing the PBS frequently. The staining results were observed using a stereo microscope and photographed for preservation.

[0118] 4. Quantification of Alizarin Red Staining (ARS) of MeGC Hydrogel Three-Dimensional Cells

[0119] Transfer each hydrogel to a 1.5 mL EP tube. Add 800 µL of 10% (v / v) acetic acid solution to each tube. Grind with a tissue grinder and vortex for 30 seconds. Sonicate at 50 W (10 s on / 2 s off) for 1 minute. Add 500 µL of mineral oil, heat in a metal bath at 85°C for 10 minutes, and then cool on ice for 5 minutes. Centrifuge at 20,000 g for 15 minutes at room temperature, then slowly aspirate 500 µL of the intermediate layer into a new centrifuge tube. Adjust the pH to 4.1-4.5 by adding 10% (v / v) ammonia solution. Pipette 150 µL into a 96-well plate and measure the absorbance at 405 nm using a microplate reader.

[0120] 2. Results

[0121] 1. CCK8 detection of hADSCs cultured in three-dimensional MeGC hydrogels encapsulating ISX-9

[0122] In a three-dimensional culture system in vitro, cells have a higher tolerance to drugs, and the safe and effective concentration ranges of drugs will change significantly. We performed CCK8 detection on hADSCs at different concentrations of ISX-9 (0, 20, 50, 100, 200, 500, 1000 μM) on days 1, 3, and 7 of three-dimensional culture. Figure 7Experimental results showed that at day 1, there was no statistically significant difference in cell viability between the ISX-9-treated and control groups. However, at days 3 and 7, cell viability decreased in the 20, 50, 100, 200, and 500 μM ISX-9-treated groups compared to the control group, with statistically significant differences (P < 0.05). The 1000 μM ISX-9 group showed the most significant decrease in cell viability, with the degree of decrease being much greater than that observed in the other ISX-9-treated groups (P < 0.05). However, it is worth noting that the 1000 μM ISX-9-treated group still exhibited robust proliferation compared to the 1-day control group. Therefore, we believe that even though 0-1000 μM is the safe concentration range for ISX-9, the high concentration of ISX-9 (1000 μM) did not produce cytotoxicity. However, to minimize the impact of altered cell viability on the osteogenic phenotype, the present invention narrowed the drug concentration to 0-600 μM.

[0123] 2. ALP staining detection in three-dimensional culture

[0124] Different concentrations of ISX-9 (50, 100, 200, 400, 600 μM) were used to encapsulate hADSCs in MeGC hydrogels. A 1 μg / mL BMP2 group was used as a positive control, and a DMSO group was used as a normal control. On the 7th day of osteogenic induction, ALP staining was analyzed ( Figure 8 ) to compare the osteogenic differentiation of ADSCs in different groups under three-dimensional culture conditions. The results showed that the ISX-9-encapsulated hydrogels expressed higher levels of ALP than the control group. Among the groups treated with different ISX-9 concentrations (50, 100, 200, 400, and 600 μM), the 100 and 200 μM ISX-9-encapsulated groups expressed significantly higher levels of ALP than the other groups. The 50, 100, 200, and 400 μM ISX-9 groups all expressed significantly higher levels of ALP than the BMP2 group, while the 600 μM ISX-9 group expressed lower levels of ALP than the BMP2 group. This indicates that in an in vitro three-dimensional culture system, ISX-9 concentrations of 50-400 μM can significantly promote ALP expression, and that 100 and 200 μM ISX-9 are the optimal concentrations for promoting ALP expression.

[0125] 3. Alizarin Red Staining and Quantification of Three-Dimensional Cultures

[0126] On the 14th day of osteogenic induction, the hydrogels were stained with alizarin red and quantitatively analyzed. Figure 9As shown, calcium deposition in the BMP2- and ISX-9-encapsulated groups increased significantly compared to the control group. The 100 μM and 200 μM ISX-9 groups exhibited significantly greater calcium deposition than the BMP2 group (P < 0.05). The 50 μM and 400 μM ISX-9 groups exhibited similar calcium deposition to the BMP2 group (P > 0.05), with no statistically significant difference. Furthermore, the 600 μM ISX-9 group exhibited lower calcium deposition than the BMP2 group. These results suggest that 100 and 200 μM ISX-9 are optimal concentrations for promoting hADSCs osteogenic differentiation in a three-dimensional culture system in vitro, providing a promising strategy for subsequent in vivo bone defect repair.

[0127] Example 4 Molecular mechanism of ISX-9 promoting osteogenic differentiation of ADSCs

[0128] To explore the molecular mechanism by which ISX-9 promotes the osteogenic differentiation of hADSCs, the present invention performed thermostable proteomic sequencing and transcriptome sequencing (RNA-seq) on hADSCs treated with 20 μM ISX-9 (ISX-9 group) and hADSCs treated with osteogenic medium without ISX-9 (control group), and performed bioinformatics analysis.

[0129] The results of thermostable proteomics are as follows Figure 10 As shown, compared with the control group, the present invention screened out four proteins with significant differences in the ISX-9 group, namely BCR, BACH1, PIGP, and DSTYK, which were annotated in the volcano plot ( Figure 10 A). Among them, the difference in BCR was the most significant. We verified these four molecules separately, and the drug affinity reaction targeted stability (DARTS) experiment found that ISX-9 could significantly reduce the proteolytic effect of proteases on BCR ( Figure 10 B), ISX-9 can increase the expression of BCR protein in hADSCs ( Figure 10 C). Knockdown of BCR in hADSCs can significantly downregulate the expression of ALP in hADSCs on the 7th day of osteogenic differentiation and significantly reduce the number of calcium nodules deposited in hADSCs on the 14th day of osteogenic differentiation ( Figure 10 D). The present invention also performed RNA-seq analysis on hADSCs treated with ISX-9. KEGG pathway enrichment analysis showed that upregulated DEGs were enriched in multiple signaling pathways related to bone metabolism, such as focal adhesion, calcium signaling, and the RAP1 signaling pathway (E). The RAP1 signaling pathway may be a direct downstream signaling pathway of the BCR protein. Therefore, ISX-9 directly targets the BCR protein and promotes the osteogenic differentiation of adipose-derived mesenchymal stem cells through the RAP1 signaling pathway.

[0130] Example 5 Effect of ISX-9-encapsulated MeGC hydrogel on regeneration and repair of skull defects in mice

[0131] 1. Materials and Methods

[0132] 1. Construction and evaluation of a non-self-healing mouse skull defect model

[0133] Six-week-old male C57BL / 6J mice were anesthetized with sodium pentobarbital (50 mg / kg), and full-thickness skull defects (3 mm in diameter) were drilled into the right parietal bone. In the blank group, the defect was left blank (no MeGC hydrogel was added). In the remaining experimental groups, different concentrations of ISX-9 were mixed with MeGC hydrogel to create hydrogel mixtures at concentrations of 50, 100, 200, and 400 μM. In the BMP2 group, BMP2 was mixed with MeGC hydrogel to create a hydrogel mixture at a concentration of 1 μg / ml. 10 μL of the hydrogel mixture was applied to the skull defect and irradiated in situ with a blue light curing lamp for 40 seconds to promote gelation. Six weeks after surgery, the mice were sacrificed and their skulls were harvested. Microcomputed tomography (μCT) was then performed to assess the in vivo osteogenic properties of ISX-9 and evaluate its ability to promote bone regeneration and repair.

[0134] 2. Microcomputed tomography

[0135] Skull samples were fixed by immersion in 4% paraformaldehyde solution for 48 hours (on a shaker at room temperature). The fixed tissues were washed twice with PBS and then stored in 75% alcohol until scanning. High-resolution tomographic images of all samples were acquired using a microCT scanner with 0.6 mm Cu and 0.5 mm Al filters (88 μA, 90 kVp). Scans were performed in high-resolution mode with a 14-minute exposure. Skull reconstructions were performed using Materialise Mimics Medical 21.0 software with a voxel resolution of 10 μm. The new bone surface area of all samples was determined using ImageJ and normalized to the original defect surface area (3 mm diameter).

[0136] 3. Statistical analysis

[0137] All data are expressed as mean ± standard deviation. SPSS 26.0 statistical software was used for independent sample t-test and one-way ANOVA analysis. Dunnett's method was used for comparison between the experimental group and the control group, and Turkey's method was used for pairwise comparison between the groups. The test level was α = 0.05, and P < 0.05 indicated statistical significance.

[0138] 2. Results

[0139] 1. μCT scanning and new bone formation analysis results

[0140] In vitro three-dimensional culture showed that 50, 100, 200, and 400 μM ISX-9 treatment had the greatest effect on promoting hADSCs osteogenic differentiation, so this concentration was selected for in vivo animal studies. In order to verify the effect of ISX-9 on promoting bone defect repair in vivo, the present invention established a non-self-healing mouse skull defect model, such as Figure 11 As shown in the results, tissue samples collected after 6 weeks showed significantly less new bone formation in the control group than in the ISX-9-treated group. ISX-9 showed the strongest ability to promote skull defect regeneration in mice when the concentration of ISX-9 in the hydrogel was 200 μM.

[0141] Example 6 Characterization of PLGA-encapsulated ISX-9-MeGC sustained-release system

[0142] ISX-9 was encapsulated using poly(lactic-co-glycolic acid) (PLGA) to prepare PLGA spheres with a particle size of approximately 260 nm. The PLGA spheres were then packaged in MeGC hydrogel to achieve sustained and slow release of ISX-9 in vivo.

[0143] By completing the preparation and partial physical characterization of ISX-9@PLGA, the particle size of ISX-9@PLGA nanospheres was found to be ~260 nm, the polydispersity index (PDI) was <0.300, and the zeta potential was -8 mV. hADSCs had a good uptake capacity for the nanospheres, with an uptake rate of approximately 92% within 24 hours ( Figure 12 ).

Claims

1. The use of ISX-9 in promoting osteogenic differentiation of mesenchymal stem cells, characterized in that: The chemical structure of ISX-9 is shown below: 。 2. Application of ISX-9 encapsulated hydrogel in promoting osteogenic differentiation of mesenchymal stem cells, characterized in that: The chemical structure of ISX-9 is shown below: 。 3. The use according to claim 1 or 2, characterized in that: The mesenchymal stem cells are selected from one or more of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells or induced pluripotent stem cells (iPSCs).

4. The use of ISX-9 in the preparation of bone injury regeneration and repair products, characterized in that: Bone damage is one or more of bone defect, bone nonunion or delayed bone healing, etc. The chemical structure of ISX-9 is shown below: 。 5. Application of ISX-9 encapsulated hydrogel in the preparation of bone injury regeneration and repair products, characterized in that: The chemical structure of ISX-9 is shown below: 。 6. The use according to claims 2 and 5, characterized in that The hydrogel is selected from one or more of methacrylate glycol chitosan (MeGC) hydrogel, gelatin hydrogel, hyaluronic acid hydrogel, and the like.

7. Application of ISX-9 sustained-release drug delivery system in the preparation of bone injury regeneration and repair products. The chemical structure of ISX-9 is shown below: 。 8. The application according to claim 7, characterized in that: The drug sustained-release delivery system includes one or more of microspheres, micelles or liposomes.

9. The use according to claims 1-9, wherein ISX-9 binds to breakpoint cluster region (BCR) protein in promoting osteogenic differentiation of mesenchymal stem cells, characterized in that: ISX-9 directly targets BCR protein and promotes osteogenic differentiation of mesenchymal stem cells through the RAP1 signaling pathway.

10. A method for inducing osteogenic differentiation of mesenchymal stem cells, characterized in that: Mesenchymal stem cells were induced to differentiate into osteoblasts using an osteogenic differentiation medium containing ISX-9, the chemical structure of which is shown below: 。

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