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

Abstract

The application provides an application of isoxazole 9 (ISX-9) in promoting mesenchymal stem cell osteogenic differentiation and bone injury regeneration repair. ISX-9 is a small molecule compound, and has the characteristics of easy to obtain, controllable structure and low cost. The in vitro experiment of the application shows that ISX-9 has the ability to induce mesenchymal stem cell osteogenic differentiation and promote the formation of mineralized nodules; the in vivo experiment shows that the hydrogel wrapped with ISX-9 can promote the regeneration repair of mouse skull defects, indicating that ISX-9 has good osteogenic induction and is a potential bone regeneration repair drug. Therefore, ISX-9 can promote mesenchymal stem cell osteogenic differentiation and be applied to bone tissue regeneration repair. The application also provides a direct action target and action mechanism of ISX-9 in promoting osteogenic differentiation. The application further provides a new drug sustained release delivery system of ISX-9, which is helpful for the application from theoretical research to clinical application.

Description

Technical Field

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

[0002] In recent years, mesenchymal stem cells (MSCs) have received increasing attention as a model for studying bone tissue regeneration and repair. Stem cells are pluripotent cells with self-renewal capacity in vivo and can differentiate into various cell types, including osteoblasts, under appropriate conditions. Bone marrow-derived mesenchymal stem cells (BMSCs) are the most widely studied cells in bone tissue engineering; however, obtaining and culturing BMSCs in vitro remains a challenging problem for clinical applications. Adipose-derived mesenchymal stem cells (ADSCs), like BMSCs, originate from the mesoderm and possess excellent osteogenic differentiation capacity. ADSCs also have the advantages of being widely available, easier to obtain, and causing less damage to the donor site. ADSCs are a preferred candidate for seed cells in future bone tissue engineering, possessing broad scientific significance and application prospects. Therefore, this invention selects ADSCs as the cell model research object. This invention is applicable to all mesenchymal stem cells derived from the mesoderm, as well as induced pluripotent stem cells (iPS cells).

[0003] Small molecule compounds, with their unique advantages such as targeted regulation of stem cell fate, controllable cost, and ease of large-scale preparation, have become the forefront of research in the field of bone regeneration. Developing a highly efficient, safe, and economical small molecule bone repair drug is an important approach to solving the clinical challenges of bone regeneration. ISX-9, composed of isoxazole and a benzene ring, has a molecular weight of 234.27 Da. Due to its small molecular weight and high lipid solubility, it can rapidly penetrate cell membranes to exert its effects. Recent studies have shown that ISX-9 exhibits significant biological activity in various cell types, particularly showing great potential in stem cell fate regulation, cell survival, and proliferation. ISX-9 not only promotes calcium influx by activating voltage-gated calcium channels and N-methyl-D-aspartate (NMDA) receptors, thus inducing neural stem cell differentiation into neurons, but it can also cross the blood-brain barrier and, depending on the activation of myocyte enhancer 2 (Mef2), promote the proliferation and neurogenesis of neural stem cells 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 crucial regulatory role in the determination of cell fate from mesoderm-derived cells. ISX-9 activates both classical and non-classical 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 lineage fate of intestinal cells 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 osteogenic differentiation of mesenchymal stem cells and regeneration and repair of bone injuries has not been reported. Summary of the Invention

[0004] The purpose of this invention is to overcome the aforementioned defects and deficiencies in the existing technology and provide the application of ISX-9 in promoting osteogenic differentiation of mesenchymal stem cells and regeneration and repair of bone injuries. This invention demonstrates that ISX-9 has the ability to induce osteogenic differentiation of mesenchymal stem cells and promote the formation of mineralized nodules; in vivo experiments show that hydrogels encapsulating ISX-9 can promote the regeneration and repair of skull defects in mice, indicating that ISX-9 has good osteogenic induction properties and is a potential drug for promoting bone regeneration and repair. Furthermore, we have discovered the mechanism and target of ISX-9 in promoting osteogenic differentiation, and simultaneously constructed an ISX-9 drug sustained-release system, overcoming the technical bottlenecks of low local delivery efficiency and short duration of action of existing hydrophobic small molecule drugs, providing a novel target and intelligent drug delivery strategy for the clinical treatment of bone injuries.

[0005] The above-mentioned objective of this invention is achieved through the following technical solution:

[0006] This invention utilizes the small molecule drug ISX-9 to investigate the effects of different concentrations of ISX-9 on the osteogenic differentiation and mineralization capacity of ADSCs. Furthermore, ISX-9 was encapsulated in a ethylene glycol-chitosan (MeGC) hydrogel developed by our research group and injected into a mouse model of non-self-healing skull defects to examine the efficacy of ISX-9 in promoting bone regeneration in vivo, providing a foundation for promoting the clinical application of ISX-9. Simultaneously, through thermostable proteomics combined with mass spectrometry and transcriptome sequencing analysis, we clarified the direct downstream targets and mechanisms of action of ISX-9. Based on the application characteristics of ISX-9, a corresponding sustained-release system was designed using the FDA-approved pharmaceutical excipient—polylactic acid-glycolic acid copolymer (PLGA)—to promote its application and commercialization.

[0007] In vitro studies showed that ISX-9 exhibits good biocompatibility within a concentration range of 5-30 μM. It upregulates the expression levels of osteogenic markers in ADSCs, such as basic granulomatase (ALP), RUNX2, osteopontin (OPN), and osteocalcin (OCN) genes and proteins. Alizarin red staining and quantification indicated that ISX-9 has the ability to induce the formation of mineralized nodules in ADSCs. The optimal osteogenic induction concentration was 20 μM, at which ISX-9's ability to promote osteogenic differentiation was superior to that of bone morphogenetic protein 2 (BMP2), a currently clinically approved drug for treating bone defects.

[0008] In vivo experimental studies showed that hydrogels encapsulating ISX-9 could promote the regeneration and repair of skull defects in mice. 6 weeks post-surgery, μCT three-dimensional reconstruction images showed that ISX-9 in the concentration range of 50-400 μM could promote the repair of skull defects in mice. Among them, 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, this invention provides the application of ISX-9 in promoting osteogenic differentiation of mesenchymal stem cells, wherein the chemical structural formula of ISX-9 is shown below: .

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

[0011] Preferably, the working concentration of ISX-9 for 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] This invention also provides the application of hydrogels encapsulating ISX-9 in promoting osteogenic differentiation of mesenchymal stem cells, wherein the chemical structural formula of ISX-9 is shown below: .

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

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

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

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

[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 pluripotent induced pluripotent stem cells (iPSCs).

[0019] This invention also provides the application of ISX-9 in the preparation of bone injury regeneration and repair products, characterized in that the bone injury is one or more of bone defects, nonunion, or delayed bone healing, and the chemical structural formula of ISX-9 is shown below: .

[0020] This invention also provides the application of hydrogels encapsulating ISX-9 in the preparation of bone injury regeneration and repair products, characterized in that the chemical structural formula of the ISX-9 is as follows: .

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

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

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

[0024] This invention also provides the application of the ISX-9 drug sustained-release delivery system in the preparation of bone injury regeneration and repair products, wherein the chemical structural formula of 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 ISX-9 directly targets the BCR protein to promote osteogenic differentiation of mesenchymal stem cells through the RAP1 signaling pathway.

[0027] This invention also provides a method for inducing osteogenic differentiation of mesenchymal stem cells, characterized in that osteogenic differentiation of mesenchymal stem cells is induced using an osteogenic differentiation-inducing medium containing ISX-9. The osteogenic differentiation-inducing medium is a conventional medium in the art (mainly containing β-glycerol phosphate and ascorbic acid), while ISX-9 is added as a promoter of osteogenic differentiation. The concentration of ISX-9 added is 15–25 μM, and the induction time is 7–14 days. The chemical structural formula 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 application of isoxazole-9 (ISX-9) in promoting osteogenic differentiation of mesenchymal stem cells and regeneration and repair of bone injuries. ISX-9 is a small molecule compound with the advantages of easy availability, controllable structure, and low cost. In vitro experiments of this invention show that ISX-9 has the ability to induce osteogenic differentiation of mesenchymal stem cells and promote the formation of mineralized nodules; in vivo experiments show that hydrogels encapsulating ISX-9 can promote the regeneration and repair of skull defects in mice, indicating that ISX-9 has good osteogenic induction and is a potential drug for promoting bone regeneration and repair. Therefore, ISX-9 can promote osteogenic differentiation of mesenchymal stem cells and can be applied to 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 helps to translate this invention from theoretical research to clinical application. Attached Figure Description

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

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

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

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

[0034] Figure 5 The results show the effect of ISX-9 on the relative expression levels of osteogenic differentiation genes in hADSCs.

[0035] Figure 6 The results show the effect of ISX-9 on the osteogenic differentiation protein levels of hADSCs.

[0036] Figure 7 The results of the CCK8 cytotoxicity assay were obtained by three-dimensional culture of ISX-9 cells in MeGC hydrogel.

[0037] Figure 8 ALP staining and quantification for three-dimensional culture of ISX-9 encapsulated in MeGC hydrogel.

[0038] Figure 9 To stain and quantify alizarin red S mineralized nodules cultured in MeGC hydrogels containing ISX-9 in three dimensions.

[0039] Figure 10 The results are for bioinformatics analysis and validation of the target of ISX-9.

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

[0041] Figure 12 Characterization of the PLGA-encapsulated ISX-9-MeGC sustained-release system. Detailed Implementation

[0042] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments 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 this technical field.

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

[0044] ISX-9, CAS No: 832115-62-5, purchased from AbMole, USA. The main research route of this invention is as follows:

[0045] Research Route 1: The role of ISX-9 in 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 DMEM containing 10% fetal bovine serum (FBS), 1% penicillin / streptomycin (P / S) antibiotics, 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 medium, and the other group was placed in complete medium containing different concentrations (5, 10, 15, 20, 25, 30, 50 μM) of ISX-9. The effect on hADSC cell viability was 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 cells, and use relevant methods and techniques to assess the osteogenic differentiation of cells.

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

[0051] Specific experimental protocol: ISX-9 or ISX-9@PLGA nanospheres at the optimal concentration for in vitro experiments were encapsulated in MeGC hydrogel and implanted into skull defects in mice. Six weeks post-operation, mice were sacrificed, and skull tissue was extracted. In vitro μCT was used to monitor bone defect healing, and CTan image processing software was used to analyze data such as trabecular volume and bone density. After decalcification, the bone tissue was paraffin-embedded, sectioned (5 µm), and subjected to histological evaluation, including H&E staining, Masson's trichrome staining, and immunohistochemistry. A three-point bending test was used to assess the biomechanical properties of the newly formed bone.

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

[0053] I. Experimental Methods

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

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

[0056] Osteogenic induced medium (OM): 10% FBS and 1% penicillin antibody were added to L-DMEM, followed by 10 mM sodium β-glycerophosphate and 50 μg / mL vitamin C. Sodium β-glycerophosphate was stored at -20 °C, and the vitamin C stock solution was prepared fresh for use immediately.

[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. Using L-DMEM medium containing 10% fetal bovine serum, 1% penicillin / streptomycin, 2 mM glutamine, 10 ng / mL fibroblast growth factor (FGF), and 1% non-essential amino acids (NEAA), ISX-9 was sequentially diluted to the corresponding working concentrations for subsequent in vitro experiments, and was prepared and used immediately.

[0059] 3. Cytotoxicity detection

[0060] hADSCs were digested with 0.25% trypsin after culture, and the complete culture medium was diluted to a cell density of 2 × 10⁻⁶. 4Cell suspension of cells / mL was seeded at 100 μL per well in a 96-well plate and incubated at 37 ℃ in 5% CO2 L-DMEM complete medium. After 1 day of incubation, the medium was replaced with L-DMEM complete medium containing 0, 5, 10, 15, 20, 25, 30, and 50 μM ISX-9, respectively. Six replicates were set for each experimental group. The medium was changed every 2 days, and days 1, 3, and 7 were designated as detection time points. At the corresponding time points, the cells were removed, the culture medium was discarded, and the cells were washed twice with sterile PBS buffer. The effect of different concentrations of ISX-9 on the proliferation of hADSCs was detected by the CCK8 assay. 90 μL of fresh culture medium and 10 μL of CCK8 reagent were added to each experimental well and the blank control well in the dark, and the cells were incubated in a cell culture incubator in the dark for 1 h. The absorbance of each experimental group's replicate wells at 450 nm was measured three times using an automated multi-functional enzyme-linked immunosorbent assay (ELISA) reader. The average values ​​were calculated, recorded, analyzed, and compared.

[0061] 4. Alizarin Red S staining and quantification

[0062] Alizarin Red S was used for qualitative analysis of the staining results of mineralized nodules, and acetic acid extraction was used for semi-quantitative analysis of the ability of ISX-9 to induce hADSCs to form mineralized nodules.

[0063] (1) Alizarin Red S staining

[0064] hADSCs were routinely digested, counted, and resuspended using 0.25% trypsin at a concentration of 3 × 10⁻⁶. 4 / well cells were seeded in 24-well plates with four replicates per group. After the cell growth density reached 50%-60%, the cell culture medium was replaced with osteogenic induction medium containing 0, 10, 15, 20, 25, 30, and 50 μM ISX-9 and BMP2 (200 ng / ml) (10 mM β-glycerophosphate sodium and 50 μg / mL vitamin C were added to the complete medium). The medium was changed every 3 days, and the medium was discarded on day 14. The cells were washed three times with PBS buffer, and 300 μL of 4% paraformaldehyde fixative was added to each well for 20 min. After fixation, each group of experimental wells was washed three times with PBS buffer. 300 μL of 1% (v / v) Alizarin Red S staining solution at pH 4.2 was added according to the product instructions. After standing at room temperature in the dark for 10 min, the staining solution was aspirated. The cells were washed repeatedly with PBS buffer until the supernatant was colorless or the color no longer changed. The cells were air-dried at room temperature and observed under a scanning electron microscope and a 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 min. Add the solution to a 1.5 mL EP tube and vortex for 30 s. Add 500 μL of mineral oil to the upper layer, heat at 85 °C for 10 min, and then place on ice for 5 min. Centrifuge at 20000×g for 15 min and aspirate 500 μL of the intermediate layer. Add 200 μL of 10% (v / v) ammonia and mix well. Transfer 150 μL to a 96-well plate and measure the absorbance at 405 nm using a microplate reader.

[0067] 5. Detection of cellular ALP activity

[0068] Add 2×10 to each well of the 24-well plate 4 hADSCs were seeded and incubated at 37°C with 5% CO2 for 24 h to allow cell adhesion. After cell adhesion, osteogenic induction medium containing different concentrations of ISX-9 (0, 10, 15, 20, 25, 30, 50 μM) and BMP2 (200 ng / ml) was added. Each group was configured with 4 replicates, and the medium was changed every 3 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, add 300 μL of 4% paraformaldehyde solution to fix the cells for 30 min; prepare the ALP staining working solution according to the ALP staining kit instructions; after fixation, wash the plate twice with PBS, add 300 μL of ALP staining working solution to each well, and stain at room temperature in the dark for 10-30 min; terminate the staining reaction with ddH2O; scan with a scanner and observe under a stereomicroscope.

[0071] (2) Quantitative detection of ALP activity

[0072] hADSCs with 2×10 4 / well cells were seeded in 24-well plates at a seeding 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 days 3 and 7, the culture medium was discarded, and the cells were washed 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 sterile EP tubes and centrifuged at 13,000 rpm, 4 °C for 10 min. 80 μL of the supernatant was collected, and the total protein content and alkaline phosphatase content were measured using a total protein quantification kit and an alkaline phosphatase assay kit, respectively. The ratio of ALP concentration to total protein concentration was calculated according to the product instructions to obtain the ALP activity for each group, expressed in U / g protein.

[0073] II. Results

[0074] 1. Cytotoxicity test results of ISX-9

[0075] The results of the CCK-8 cytotoxicity assay showed that ( Figure 1 Compared with the control group, at 1 day, the culture medium containing ISX-9 did not affect 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 statistically significant 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 difference was not statistically significant (P>0.05), while the OD values ​​of the 25 and 30 μM ISX-9 groups were lower than those of the control group, indicating lower cell viability, but the difference was 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, with a statistically significant difference (P<0.05). Therefore, low concentrations of ISX-9 (5-30 μM ISX-9) can promote the proliferation of hADSCs. ISX-9 (μM) showed no cytotoxicity to hADSCs, and even at 3 days, low concentrations of ISX-9 enhanced the proliferation of hADSCs.

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

[0077] Figure 2 , 3 The results of ALP staining and quantification were presented on days 3 and 7 of osteogenic induction. The results showed that on day 3, ALP staining in the positive control group (BMP2) was darker than in the control group. ALP staining in all ISX-9 treatment groups was darker than in the BMP2 group, with the 20 μM ISX-9 group showing the deepest ALP staining. The trend of ALP activity quantification was consistent with the staining results. The ALP activity value in the BMP2 group was approximately twice that of the control group. The 10 and 50 μM ISX-9 treatment groups were significantly higher than the control group, but essentially consistent with the BMP2 group, with no statistical significance (P > 0.05). However, the 15, 20, 25, and 30 μM ISX-9 treatment groups showed a significant increase compared to the BMP2 group, with statistical differences (P < 0.05). The trends of ALP staining and quantification results at 7 days were basically the same as those at 3 days. The ALP expression levels in the 15, 20, 25, and 30 μM ISX-9 treatment groups were significantly higher than those in the control group and the positive control group BMP2 group, with statistical differences (P < 0.05). From the qualitative staining and quantitative activity detection results of ALP at 3 days and 7 days, different concentrations of ISX-9 could promote ALP expression in hADSCs, with 15-25 μM being the optimal concentration range and 20 μM being the optimal drug concentration.

[0078] 3. Results of staining and semi-quantitative analysis of mineralized nodules

[0079] hADSCs were co-cultured with ISX-9 for 14 days and then stained with Alizarin Red S. The results are as follows: Figure 4 As shown, at safe drug concentrations, the ISX-9 group formed more and larger mineralized nodules compared to the control group, according to the semi-quantitative analysis results of alizarin red ( Figure 4 The results showed that the 20 μM ISX-9 group had the highest mineralization. The 15, 20, 25, and 30 μM ISX-9 concentrations significantly promoted hADSC formation more than the control and BMP2 groups, with statistically significant differences (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. Based on the combined results of Alizarin Red S qualitative staining and semi-quantitative analysis of the mineralized nodules, the 15-25 μM ISX-9 concentration showed the best ability to induce hADSC mineralized nodule formation, with 20 μM being the optimal osteogenic drug concentration.

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

[0081] I. Experimental Methods

[0082] 1. The relative expression levels of related genes during osteogenic differentiation of hADSCs were detected by reverse transcription-qPCR.

[0083] (1) Total RNA extraction using the Trizol method

[0084] After discarding the old culture medium and washing with PBS, add 500 μL of Trizol lysis buffer to each well of a 24-well plate, thoroughly lyse the cells 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 inverting, and incubate at room temperature for 3 min. Centrifuge at 12000×g, 4 °C for 15 min, and carefully transfer the supernatant to a 1.5 mL enzyme-free centrifuge tube. Add an equal volume of anhydrous ethanol to the tube, mix by pipetting, and transfer the sample to an RNA centrifuge column. Centrifuge at 4000×g for 1 min and discard the waste liquid. Add 500 μL of WashBuffer 1 to the column, centrifuge at 12000×g, 4 °C for 1 min, and discard the waste liquid. Add 500 μL of WashBuffer 2 to the column, centrifuge at 12000×g, 4 °C for 1 min, and discard the waste liquid. Return the centrifuge column to the collection tube and centrifuge at 12000×g, 4 °C for 1 min to thoroughly remove any residual waste liquid. Transfer the centrifuge column to a new 1.5 mL RNase-free centrifuge tube and allow it to air dry for 2 min. Add 20 μL of RNase-free water to the centrifuge column, incubate at room temperature for 2 min, and then centrifuge at 12000×g, 4 °C for 1 min to obtain total RNA.

[0085] RNA concentration was detected using a NanaDrop micro-volume spectrophotometer. 1 μL of extracted total RNA was taken, and the concentration was measured three times, with the average value recorded. RNA purity was determined by the ratio of the OD values ​​at 260 nm and 280 nm. A ratio between 1.8 and 2.0 indicated high total RNA purity, meeting the requirements for RT-qPCR.

[0086] Prepare a 20 μL reaction mixture according to the TAKARA kit instructions, ensuring a consistent RNA concentration in the samples. Then, perform reverse transcription on a standard PCR instrument (37 °C for 15 min, 85 °C for 5 s) to synthesize cDNA.

[0087] (2) Quantitative real-time PCR (qPCR)

[0088] 1) Primer design

[0089] Primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. All primer sequences involved in this Real-Time PCR experiment are shown in Table 2.

[0090] 2) System

[0091] According to the Novizan kit instructions, prepare a 20 μL reaction system for qPCR detection as shown in the table below.

[0092] Table 1 qPCR reaction system

[0093]

[0094] 3) qPCR experimental reaction conditions: PCR reaction was performed in a real-time quantitative PCR instrument, and the reaction program was set as follows: pre-denaturation (95 ℃, 30 s); denaturation (95 ℃, 10 s); annealing and extension (60 ℃, 30 s), 40 cycles.

[0095] 4) Analysis of Real-time PCR Experiment Results: Three replicates were set up for the same sample (technical replication). The Ct values ​​of each sample in the control group and each experimental group were measured, and the relative expression level of the target gene was calculated. The experiment was repeated 3 times (experimental replication).

[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 protein; OSX: Osteoblast-specific transcription factor; OPN: Osteopontin; OCN: Osteocalcin.

[0099] 2. Western blot detection

[0100] 1x10⁻⁶ seeds were seeded in a 12-well plate. 5 After 24 hours, hADSCs were processed, and the culture medium was discarded. Four replicates were set up for each group, and the medium was replaced with osteogenic mineralization induction medium containing different concentrations of ISX-9 and BMP2. The medium was changed every 3 days. Samples were collected on day 7, the original culture medium was discarded, and the samples were washed three times with PBS buffer. The PBS in the wells was aspirated as dry as possible. RIPA lysis buffer containing PMSF and a protease phosphatase inhibitor was prepared. 100-120 μL of RIPA lysis buffer was added to each sample well, and the cells were lysed on ice for 30 min. The lysate was collected using a cell scraper into EP tubes. The cells were pre-chilled to 4 °C and centrifuged at 13,000 rpm for 15 min. The bottom precipitate was discarded, and the supernatant was collected. The total protein concentration was determined and balanced using a BCA kit. An appropriate amount of loading buffer was added, and the mixture was incubated in a 100 °C water bath or metal bath for 10 min. The mixture was then transferred to a -80 °C freezer for storage.

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

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

[0103] Membrane blocking and antibody incubation: After transfer, the NC membrane was blocked with pre-prepared 5% skim milk on a shaker at room temperature for 1 h. After blocking, the membrane was rinsed with TBST for 5 min, the rinse buffer was discarded, the membrane was aspirated dry, and the primary antibody for the target protein, prepared according to the instructions, was added for incubation overnight on a shaker at 4 °C. After primary antibody incubation, the bands were rinsed three times with TBST on a shaker at room temperature for 10 min each time. Secondary antibody was prepared with 5% skim milk and added to the incubation chamber, and incubated on a shaker at room temperature for 1 h. After secondary antibody incubation, the membrane was rinsed three times with TBST on a shaker at room temperature for 10 min each time. ECL chemiluminescence developing solution was prepared, and the NC membrane containing the target protein was immersed in the solution for 1 min under light-protected conditions. The membrane was removed with flat-tipped forceps, excess chemiluminescence solution was aspirated, and the membrane was developed using a developer. The bands were qualitatively observed and recorded. The bands on the membrane were quantitatively analyzed and compared using ImageJ image processing software.

[0104] II. Results

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

[0106] The relative expression levels of osteogenic differentiation genes in hADSCs were detected using qPCR. RUNX2 was detected on day 3 of osteogenic differentiation, OPN on day 7, and OCN on day 14. Results are as follows: Figure 5As shown: Regarding RUNX2 gene expression, compared with the control group, RUNX2 was highly expressed in the BMP2 group and the treatment groups with different concentrations of ISX-9, and the difference was statistically significant (P < 0.05). The RUNX2 expression in the 15, 20, 25, and 30 μM ISX-9 groups was significantly higher than that in the BMP2 group, and the difference was statistically significant (P < 0.05). The RUNX2 gene expression in the 10 and 50 μM ISX-9 groups was slightly lower than that in the BMP2 group, and the difference was statistically significant (P > 0.05). Regarding OPN gene expression, compared with the control group, OPN expression was significantly higher in the BMP2 group and the treatment groups with different concentrations of ISX-9 (P < 0.05). There was no significant difference in relative OPN gene expression between the 10 and 30 μM ISX-9 groups and the control group (P > 0.05). OPN expression was significantly higher in the 15, 20, and 25 μM ISX-9 groups than in the BMP2 group, while OPN expression was slightly lower in the 50 μM ISX-9 group than in the BMP2 group (P < 0.05). At 14 days, compared with the control group, OCN expression was significantly higher in the BMP2 group and the treatment groups with different concentrations of ISX-9 (P < 0.05). OCN gene expression was significantly higher in the 15, 20, 25, and 30 μM ISX-9 groups than in the BMP2 group (P < 0.05). The expression of OCN gene in the ISX-9 group was basically the same as that in the BMP2 group, with no statistically significant difference. In summary, the qPCR results indicate that, in terms of promoting the expression levels of genes related to osteogenic differentiation of hADSCs, ISX-9 at safe concentrations can promote the expression of osteogenic differentiation marker genes in hADSCs, with 20 μM ISX-9 showing the strongest effect in promoting osteogenic differentiation of hADSCs.

[0107] 2. Western blot results

[0108] Western blot results are as follows Figure 6 As shown, we detected the protein levels of key transcription factors RUNX2 and OSX on day 7 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: MeGC hydrogel encapsulating ISX-9 promotes osteogenic differentiation of hADSCs

[0110] To better simulate the three-dimensional growth environment of cells in vivo, this study used MeGC hydrogel to embed hADSCs and loaded ISX-9 onto the MeGC hydrogel to explore the ability of ISX-9 to promote osteogenic differentiation of ADSCs in a three-dimensional growth environment. This also provides a reference for the safe and effective drug concentration range in the in vivo model.

[0111] I. Experimental Methods

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

[0113] Dissolve the MeGC hydrogel lyophilized powder in PBS at a weight / volume percentage of 4%. Digest ADSCs cells with trypsin, add an equal volume of complete culture medium to stop digestion, transfer the cell culture to a 15 mL centrifuge tube, centrifuge at 180 g for 3 min, and collect the cell pellet. Repeat the process at 2 × 10⁻⁶. 6 ADSCs hydrogel suspensions were prepared using 2% MeGC hydrogel, 6 μM riboflavin, and appropriate drug concentrations. 40 μL of the well-mixed hydrogel was dropped onto a polytetrafluoroethylene (PTFE) plate and irradiated with a blue light curing lamp (Carent LY-A180) for 40 s. The cured hydrogel was then transferred to uncoated 48-well plates, and 800 μL of complete culture medium was added to each well. The plates were incubated in a cell culture incubator, with medium changes performed every 2-3 days. On days 1, 3, and 7 of culture, the effect of different concentrations of ISX-9 on hADSC proliferation was assessed using the CCK-8 assay. 900 μL of fresh culture medium and 100 μL of CCK-8 reagent were added sequentially to each experimental well and the blank control well in the dark. The wells were incubated in the dark for 3 h. The absorbance of each experimental group was measured three times at 450 nm using an automated multi-functional enzyme-linked immunosorbent assay (ELISA) reader. The average values ​​were calculated, recorded, analyzed, and compared.

[0114] 2. MeGC hydrogel three-dimensional cell alkaline phosphatase (ALP) staining

[0115] MeGC hydrogels induced for osteogenic formation for 7 days were washed twice with PBS. Each hydrogel was fixed with 300 µL of 4% paraformaldehyde for 20 min. ALP staining working solution was prepared according to the ALP staining kit instructions, and 300 µL of the solution was added. The mixture was then incubated at 37 °C in the dark. The total reaction time depended on the reaction effect. To terminate the reaction, the ALP staining working solution was completely aspirated, and the mixture was washed twice with ddH2O. The staining results were observed and photographed using a stereomicroscope.

[0116] 3. MeGC hydrogel three-dimensional cellular alizarin red staining (ARS)

[0117] MeGC hydrogels induced for osteogenic formation for 14 days were washed twice with PBS. Each hydrogel was fixed with 300 µL of 4% paraformaldehyde for 20 min. 300 µL of alizarin red staining solution was added, and the mixture was reacted at room temperature for 10 s. After washing with PBS on a shaker for 24 h to remove excess stain, the PBS was changed several times during the process. The staining results were observed and photographed using a stereomicroscope.

[0118] 4. Quantitative analysis of three-dimensional alizarin red (ARS) staining of MeGC hydrogel 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, homogenize with a tissue homogenizer, and vortex for 30 s. Sonicate at 50 W for 1 min (10 s on, 2 s off). Add 500 µL of mineral oil, heat in a metal bath at 85 °C for 10 min, then cool on ice for 5 min. Centrifuge at 20,000 g for 15 min at room temperature, then slowly aspirate 500 µL of the intermediate layer to a new centrifuge tube. Adjust the pH to 4.1-4.5 by adding 10% (v / v) ammonia, and aspirate 150 µL to a 96-well plate. Measure the absorbance at 405 nm using a microplate reader.

[0120] II. Results

[0121] 1. CCK8 assay of hADSCs cultured in three dimensions using MeGC hydrogels encapsulated with ISX-9

[0122] Cells in an in vitro three-dimensional culture system exhibit higher tolerance to drugs, and both the safe and effective concentration ranges of the drugs change significantly. We analyzed hADSCs at different concentrations of ISX-9 (0, 20, 50, 100, 200, 500, and 1000 μM) on days 1, 3, and 7 of three-dimensional culture using CCK8 assays. Figure 7The experimental results showed that at day 1, there was no statistically significant difference in cell viability between the ISX-9 treatment group and the control group. However, at days 3 and 7, compared with the control group, cell viability decreased in all four ISX-9 treatment groups (20, 50, 100, 200, and 500 μM), with statistically significant differences (P < 0.05). The 1000 μM group showed the most significant decrease in cell viability, far exceeding the decrease in other ISX-9 concentrations, with statistically significant differences (P < 0.05). However, it is noteworthy that the 1000 μM ISX-9 treatment group still exhibited stronger proliferative activity compared to the control group at day 1. Therefore, we believe that even though 0-1000 μM is the safe concentration range for ISX-9, high concentrations of ISX-9 (1000 μM) did not produce cytotoxicity. However, to reduce the impact of altered cell viability on osteogenic phenotype, this invention reduces the drug application 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 encapsulated with equal volumes of hADSCs in MeGC hydrogels. A 1 μg / mL BMP2 group was used as a positive control, and a DMSO group as a normal control. On day 7 of osteogenic induction, ALP staining was analyzed. Figure 8 This study aimed to compare the osteogenic differentiation of ADSCs in different groups under three-dimensional culture conditions. The results showed that ALP expression levels in hydrogels embedded with ISX-9 were higher than in the control group. Among the groups with different concentrations of ISX-9 (50, 100, 200, 400, and 600 μM), the ALP expression levels in the 100 and 200 μM ISX-9 groups were significantly higher than in the other groups. The ALP expression levels in the 50, 100, 200, and 400 μM ISX-9 groups were all significantly higher than in the BMP2 group, while the ALP expression level in the 600 μM ISX-9 group was lower than in the BMP2 group. This indicates that under in vitro three-dimensional culture conditions, 50-400 μM ISX-9 can significantly promote ALP expression, and that 100 and 200 μM ISX-9 are the optimal drug concentrations for promoting ALP expression.

[0125] 3. Alizarin Red staining and quantification in three-dimensional culture

[0126] On day 14 of osteogenic induction, the hydrogel was stained with alizarin red and quantitatively analyzed. Results are as follows: Figure 9As shown, the calcium deposition in the groups embedded with BMP2 and ISX-9 was significantly increased compared to the control group. The calcium deposition in the 100 μM and 200 μM ISX-9 groups was significantly higher than that in the BMP2 group (P < 0.05). The calcium deposition in the 50 μM and 400 μM ISX-9 groups was basically the same as that in the BMP2 group (P > 0.05). The calcium deposition in the 600 μM ISX-9 group was lower than that in the BMP2 group. These results indicate that, in an in vitro three-dimensional culture system, 100 and 200 μM ISX-9 are the optimal drug concentrations for promoting osteogenic differentiation of hADSCs, providing a reference for subsequent drug applications in in vivo bone defect repair.

[0127] Example 4: Molecular mechanism by which ISX-9 promotes osteogenic differentiation of ADSCs

[0128] To explore the molecular mechanism by which ISX-9 promotes osteogenic differentiation of hADSCs, this invention performed thermostable proteomics 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 then performed bioinformatics analysis.

[0129] Thermostable proteomics results such as Figure 10 As shown, compared with the control group, this invention screened out four proteins with significant differences in the ISX-9 group: BCR, BACH1, PIGP, and DSTYK, which are annotated in the volcano diagram. Figure 10 A). Among them, the difference in BCR was the most significant. We validated these four molecules separately, and the Drug Affinity Response Target Stability (DARTS) experiment showed that ISX-9 could significantly reduce the proteolytic effect of streptase on BCR (A). Figure 10 B), ISX-9 can increase the expression of BCR protein in hADSCs (B), Figure 10 C). Knocking down BCR in hADSCs significantly downregulated ALP expression in hADSCs on day 7 of osteogenic differentiation and significantly reduced the number of calcium nodules deposited in hADSCs on day 14 of osteogenic differentiation. Figure 10 (D) This invention also performed RNA-seq analysis on ISX-9 treated hADSCs. KEGG pathway enrichment analysis showed that the upregulated DEGs were enriched in multiple signaling pathways related to bone metabolism, such as focal adhesion, calcium signaling, and the RAP1 signaling pathway (E). Among them, the RAP1 signaling pathway may be a direct downstream signaling pathway of the BCR protein. Therefore, ISX-9 directly targets the BCR protein to promote osteogenic differentiation of adipose-derived mesenchymal stem cells through the RAP1 signaling pathway.

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

[0131] I. Materials and Methods

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

[0133] Six-week-old male C57BL / 6J mice were anesthetized with sodium pentobarbital (50 mg / kg). A full-thickness skull defect (3 mm in diameter) was drilled from the right parietal bone. In the control group, the defect was left empty (without MeGC hydrogel). In the other experimental groups, different concentrations of ISX-9 were mixed with MeGC hydrogel to prepare hydrogel mixtures with concentrations of 50, 100, 200, and 400 μM. In the BMP2 group, BMP2 was mixed with MeGC hydrogel to prepare a hydrogel mixture with a concentration of 1 μg / ml. 10 μL of each hydrogel mixture was added to the skull defect, and the mice were irradiated in situ with a blue light curing lamp for 40 seconds to promote gelation. Six weeks post-surgery, the mice were sacrificed, and their skulls were harvested. Micro-computed 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 soaking in 4% paraformaldehyde solution for 48 h (on a shaker at room temperature). The fixed tissues were washed twice with PBS and then stored in 75% ethanol for later scanning. High-resolution tomographic images of all samples were acquired using μCT scanning. 0.6 mm Cu and 0.5 mm Al filters were used (88 μA, 90 kVp). Scans were performed at high resolution for 14 min, and skull reconstruction was performed using Materialise Mimics Medical 21.0 software with a voxel resolution of 10 μm. The neobone surface area of ​​all samples was measured using ImageJ and normalized to the original defect surface area (3 mm in diameter).

[0136] 3. Statistical Analysis

[0137] All data are expressed as mean ± standard deviation. Independent samples t-tests and one-way ANOVA analyses were performed using SPSS 26.0 statistical software. Dunnett's method was used for comparisons between experimental and control groups, and Turkey's method was used for pairwise comparisons between groups. The significance level was set at α = 0.05, and p < 0.05 was considered statistically significant.

[0138] II. Results

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

[0140] In vitro three-dimensional culture revealed that treatments with 50, 100, 200, and 400 μM ISX-9 showed the greatest effect in promoting osteogenic differentiation of hADSCs. Therefore, this invention selected these concentrations for in vivo animal studies. To verify the in vivo effect of ISX-9 in promoting bone defect repair, this invention used a mouse model of non-self-healing skull defects, such as... Figure 11 As shown, when tissue samples were taken 6 weeks later, the area and volume of new bone formation in the control group were significantly less than those in the ISX-9 treated group. The ISX-9 concentration in the hydrogel was 200 μM, which demonstrated the strongest ability of ISX-9 to promote the regeneration of skull defects in mice.

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

[0142] ISX-9 was encapsulated with polylactic-co-glycolic acid copolymer (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] Through the preparation and partial physical characterization of ISX-9@PLGA, the particle size (~260 nm), polydispersity index (PDI) (<0.300), and zeta potential (-8 mV) of ISX-9@PLGA nanospheres were determined. hADSCs exhibited good uptake capacity of the nanospheres, with an uptake rate of approximately 92% after 24 hours. Figure 12 ).

Claims

1. The application of ISX-9 in the preparation of drugs that promote osteogenic differentiation of mesenchymal stem cells, characterized in that, The chemical structural formula of ISX-9 is shown below: The mesenchymal stem cells are selected from one or more of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, and adipose mesenchymal stem cells, and the osteogenic differentiation is used for the repair of bone defects, nonunion, or delayed bone healing.

2. The application of hydrogels encapsulating ISX-9 in the preparation of drugs promoting osteogenic differentiation of mesenchymal stem cells, characterized in that... The chemical structural formula of ISX-9 is shown below: The mesenchymal stem cells are selected from one or more of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, and adipose mesenchymal stem cells, and the osteogenic differentiation is used for the repair of bone defects, nonunion, or delayed bone healing.

3. A method for inducing osteogenic differentiation of mesenchymal stem cells in vitro, characterized in that, Mesenchymal stem cells were induced to undergo osteogenic differentiation using an osteogenic differentiation-inducing medium containing ISX-9. ISX-9 was added as a promoter of osteogenic differentiation at a concentration of 15–25 μM, and the induction time was 7–14 days. The chemical structural formula of ISX-9 is shown below: The mesenchymal stem cells are selected from one or more of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, and adipose mesenchymal stem cells.

4. The application of ISX-9 in the preparation of drugs for bone injury regeneration and repair, characterized in that, Bone injury includes one or more of bone defects, nonunion, or delayed bone healing. The chemical structural formula of ISX-9 is shown below: 。 5. The application of hydrogels encapsulating ISX-9 in the preparation of drugs for bone injury regeneration and repair, characterized in that, The chemical structural formula of ISX-9 is shown below: The bone injury is one or more of bone defects, nonunion, or delayed bone healing.

6. The application according to claim 2 or 5, characterized in that, The hydrogel is selected from one or more of ethylene glycol chitosan hydrogel, gelatin hydrogel, or hyaluronic acid hydrogel.

7. The application of the ISX-9 drug sustained-release delivery system in the preparation of drugs for bone injury regeneration and repair, wherein the chemical structural formula of ISX-9 is shown below: The bone injury is one or more of bone defects, nonunion, or delayed bone healing.

8. The application according to claim 7, characterized in that, Drug sustained-release delivery systems include one or more of microspheres, micelles, or liposomes.

Images