Method for preparing functionalized bone marrow mesenchymal stem cell exosome through mechanical stimulation

Abstract

The invention relates to the technical field of exosomes, and particularly discloses a method for preparing functionalized bone marrow mesenchymal stem cell exosomes through mechanical stimulation, which comprises the following steps: extracting bone marrow mesenchymal stem cells (BMSCs); periodic stretching mechanical stimulation with the intensity of 10% is applied to the BMSCs; the method comprises the following steps: collecting a cell supernatant, filtering the cell supernatant by 0.22 mu m, and extracting an exosome by an ultracentrifugation method to obtain a functionalized exosome MS-Exo; the preparation of the mesenchymal stem cell exosome is optimized through mechanical stimulation, the biological activity of the exosome is remarkably improved, proliferation and differentiation of bone cells are promoted, and the progress of steroid-induced femoral head necrosis is slowed down. A research result shows that the exosome subjected to mechanical stimulation shows a good osteogenesis effect in vitro and in vivo, and the structure and the function of bone tissues can be effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of exosome technology, specifically relating to a method for preparing functionalized bone marrow mesenchymal stem cell exosomes through mechanical stimulation. Background Technology

[0002] Hormone-induced avascular necrosis of the femoral head is an ischemic necrosis of bone tissue caused by long-term use of hormone medications. It primarily affects the blood supply to the femoral head, leading to osteocyte death and bone structure destruction. Current treatments include conservative and surgical approaches, but these methods often fail to effectively halt disease progression. In recent years, researchers have begun to focus on the application of stem cells and their exosomes in bone regeneration, especially bone marrow mesenchymal stem cells, which have attracted widespread attention due to their excellent osteogenic capacity.

[0003] Currently, stem cell therapy has made some progress in the research of osteonecrosis. Bone MSCs can secrete a variety of bioactive factors that promote the repair and regeneration of bone tissue. However, traditional stem cell therapy methods suffer from problems such as low cell survival rate and poor post-transplantation integration. In recent years, exosomes, as important mediators of intercellular communication, have been increasingly recognized for their important role in promoting cell growth, migration, and differentiation. Enhancing exosome secretion from BMSCs through mechanical stimulation may provide a new approach for the treatment of hormone-induced osteonecrosis of the femoral head.

[0004] Despite the promising potential of stem cells and their exosomes in bone regeneration, current research faces several challenges. First, effectively obtaining and preparing functionalized exosomes remains a technical hurdle. Second, the bioactivity and osteogenic effects of exosomes in vivo have not been fully validated. Furthermore, existing treatments often fail to intervene in the early stages of the disease, leading to patients receiving treatment at a more advanced stage. Therefore, a novel treatment approach is urgently needed to effectively intervene in the early stages of hormone-induced avascular necrosis of the femoral head, slowing disease progression and protecting the structure and function of the femoral head. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing functionalized bone marrow mesenchymal stem cell exosomes through mechanical stimulation, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for preparing functionalized bone marrow mesenchymal stem cell exosomes by mechanical stimulation includes the following steps:

[0008] (1) Extraction of bone marrow mesenchymal stem cells (BMSCs);

[0009] (2) Apply periodic tensile mechanical stimulation with an intensity of 10% to BMSCs;

[0010] (3) Collect the cell supernatant, filter it through 0.22 μm, and extract exosomes by ultracentrifugation to obtain functionalized exosomes MS-Exo.

[0011] Preferably, the mechanical stimulation is uniaxial tension with a frequency of 0.5–2 Hz and a duration of 6–48 hours.

[0012] Preferably, the BMSCs are identified as having a CD90 and CD29 positive expression rate of ≥95%, a CD45 negative expression rate of ≤2%, and the ability to differentiate into osteoblasts, chondrocytes, and adipocytes.

[0013] A functionalized bone marrow mesenchymal stem cell exosome, prepared by any of the methods described above, wherein the exosome has a particle size of 100–200 nm, is positive for the surface marker TSG101 and negative for Calnexin;

[0014] Preferably, the exosomes contain active ingredients that regulate the Wnt / β-catenin signaling pathway, including upregulation of TCF7, β-catenin, LRP7 expression and / or downregulation of GSK-3β expression.

[0015] Application of a functionalized exosome in the preparation of a drug for treating hormone-induced avascular necrosis of the femoral head.

[0016] A pharmaceutical formulation for treating hormone-induced avascular necrosis of the femoral head, comprising the aforementioned functionalized exosomes and carrier;

[0017] Preferably, the concentration of the exosomes is 50–200 μg / mL;

[0018] Preferably, the carrier is physiological saline or phosphate buffer.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] This invention optimizes the preparation of bone marrow mesenchymal stem cell exosomes through mechanical stimulation, significantly improving the bioactivity of exosomes, promoting osteocyte proliferation and differentiation, and slowing the progression of hormone-induced avascular necrosis of the femoral head. The results show that mechanically stimulated exosomes exhibit good osteogenic effects both in vitro and in vivo, effectively improving the structure and function of bone tissue, providing a new and effective method for the early treatment of hormone-induced avascular necrosis of the femoral head, and showing promising clinical application prospects. Attached Figure Description

[0021] Figure 1This is a schematic diagram illustrating the morphological observation, multi-lineage differentiation capacity verification, and phenotypic identification of BMSCs in Example 2 of the present invention; it includes: A-cell morphology; B-osteogenic differentiation staining; C-chondrogenic differentiation staining; D-adipogenic differentiation staining; E-flow cytometry phenotypic analysis.

[0022] Figure 2 This is a schematic diagram of the dexamethasone (Dex) inhibitory concentration screening and the rescue effect of mechanically stimulated conditioned medium (BMSC-CM) in Example 3 of the present invention; it includes: A-Dex concentration gradient proliferation inhibition; B-CCK-8 proliferation detection; CD-EdU cell proliferation detection; EF-osteoblast gene expression RT-PCR;

[0023] Figure 3 This is a schematic diagram illustrating the exosome characterization and its rescue effect on Dex inhibition in Embodiment 4 of the present invention; it includes: A-TEM morphological observation; B-NTA particle size distribution; C-WB marker detection; D-cell uptake verification; E-CCK-8 proliferation detection; FG-EdU proliferation detection;

[0024] Figure 4 This is a schematic diagram illustrating the promoting effect of MS-Exo on osteogenic differentiation and mineralization of MC3T3-E1 under Dex inhibition environment in Example 5 of the present invention; it includes: AB-ALP staining and quantification; CD-ARS mineralized nodule staining and quantification; EI-osteogenic gene qPCR analysis;

[0025] Figure 5 This is a schematic diagram of principal component analysis (PCA) and differentially expressed gene screening in Embodiment Six of the present invention; it includes: A - PCA distribution between groups; B - differentially expressed genes between Dex and Control groups; differentially expressed genes between CM group and D group; D - Venn diagram of common differentially expressed genes; E - gene expression heatmap;

[0026] Figure 6 This is a schematic diagram of GO functional annotation and KEGG pathway enrichment analysis of differentially expressed genes in Example 6 of the present invention; it includes: AB-GO enrichment analysis; CD-KEGG pathway enrichment; and changes in the expression of the key gene TCF7 in the EF-Wnt pathway.

[0027] Figure 7 This is a schematic diagram of the Wnt / β-catenin pathway mechanism in Example 6 of the present invention; it includes: A-β-catenin immunofluorescence nuclear translocation; BD-Wnt pathway gene qPCR; and E-GSK-3β expression inhibition.

[0028] Figure 8This is a schematic diagram illustrating the verification of osteogenic function after Dkk-1 blockade in Example 7 of the present invention; it includes: AB-ALP staining and quantification; CD-ARS staining and quantification; EH-osteogenic gene qPCR;

[0029] Figure 9 This is a Micro-CT schematic diagram of the mechanical stimulation-functionalized exosomes used to treat SONFH rats according to the present invention. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1:

[0032] Please see Figures 1-9 As shown, a method for preparing functionalized bone marrow mesenchymal stem cell exosomes via mechanical stimulation includes:

[0033] (1) Extraction of bone marrow mesenchymal stem cells (BMSCs):

[0034] Bone marrow from the femur of 6-week-old SD rats was washed with α-MEM medium containing 10% fetal bovine serum and collected by centrifugation (1200 rpm, 5 min).

[0035] Inoculate into culture dishes and culture at 37°C and 5% CO2. Change the medium every 3 days and subculture to P3 for later use.

[0036] (2) Apply periodic tensile mechanical stimulation of 10% intensity to BMSCs:

[0037] P3 generation BMSCs were seeded on Flexcel I cell mechanical loading plates and subjected to uniaxial periodic tension (intensity 10%, frequency 1Hz) for 24 hours.

[0038] (3) The cell supernatant was collected, filtered through a 0.22 μm filter, and exosomes were extracted by ultracentrifugation to obtain functionalized exosomes MS-Exo:

[0039] Collect the cell supernatant and filter it through a 0.22 μm filter membrane;

[0040] The ultracentrifugation process was carried out sequentially at 4℃, 300g×10min, 2000g×20min, 10,000g×30min, and 100,000g×70min (precipitate was MS-Exo), resuspended in PBS, and stored at -80℃.

[0041] Example 2:

[0042] Mice were soaked in 75% ethanol for 5 minutes. Femurs and tibias were separated under aseptic conditions, soft tissues were removed, and the bone marrow cavity was rinsed with PBS containing 1% penicillin and antibiotics until the bone turned white. The bone marrow rinsing fluid was collected, filtered through a 70 μm cell sieve, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in DMEM medium containing 10% FBS. The cells were seeded in culture dishes and cultured in a 37°C, 5% CO2 incubator. The medium was changed for the first time after 72 hours to remove non-adherent cells, and thereafter every 3 days.

[0043] like Figure 1 As shown in (A), the inverted microscope image reveals that BMSCs have a typical spindle morphology.

[0044] Verify its multi-directional differentiation ability:

[0045] Osteogenic differentiation: Osteogenic induction medium was used, with the medium changed every 3 days for 21 days. The mineralized nodules were observed after staining with Alizarin Red for 10 minutes.

[0046] Chondrogenic differentiation: The microsphere culture method was used in centrifuge tubes, and chondrogenic induction medium was used for 21 days. The cartilage matrix was observed after staining with Alcian blue for 30 minutes.

[0047] Adipogenic differentiation: Adipogenic induction medium was used, with the medium changed every 3 days for 14 days. The lipid droplets were observed after staining with Oil Red O for 15 minutes.

[0048] like Figure 1 As shown in (BD), in osteogenic, chondrogenic and adipogenic differentiation assays, induced BMSCs exhibit mineralized nodules, cartilage tissue and lipid droplets.

[0049] Phenotypic identification by flow cytometry: Third-generation cells were collected, digested with 0.25% trypsin, washed with PBS, resuspended in 100 μL PBS, and antibody was added. The cells were incubated in the dark for 30 minutes (4°C). Results were as follows: Figure 1 As shown in (E), mesenchymal stem cell markers CD90 and CD29 are positive, but CD45 is negative;

[0050] As can be seen from the above, BMSCs possess the basic characteristics of mesenchymal stem cells.

[0051] Example 3:

[0052] Screening for Dexamethasone (Dex) inhibitory concentrations:

[0053] MC3T3-E1 at 5×10 3Cells were seeded at a density of 1 / well in 96-well plates and cultured at 37°C and 5% CO2 for 24 hours until adherence. Dex concentration gradients were set up: 0 (Control), 1, 5, 25, 50, 100, 200, 300 μM. Fresh medium containing the corresponding concentration of Dex was added to each well (100 μL / well). Each group had 6 replicates and was cultured for 24 hours.

[0054] CCK-8 assay for proliferation: Add 10 μL of CCK-8 solution to each well, incubate at 37°C in the dark for 2 hours, and measure the absorbance (OD value) at 450 nm using a microplate reader. Figure 2 As shown in (A), treatment with 50 μM Dex for 24 hours significantly inhibited proliferation, and this concentration was used in subsequent experiments.

[0055] Preparation of biomechanical conditioned medium (BMSC-CM):

[0056] BMSCs were inoculated into stretchable silicone culture plates and cultured in serum-free medium for 24 hours for synchronization. The stretching mode was uniaxial periodic stretching with intensity gradients of 5%, 10%, and 15%; the frequency was 1 Hz (60 cycles / min) and the duration was 24 hours.

[0057] After stimulation, the cell supernatant was collected, centrifuged at 1200 rpm for 10 minutes to remove cell debris, and the supernatant was filtered through a 0.22 μm filter for sterilization, aliquoted and stored at -80℃.

[0058] Verification of the rescue effect of BMSC-CM on Dex inhibition:

[0059] CCK-8 proliferation assay: MC3T3-E1 cells were inoculated into 96-well plates, and the corresponding culture medium was added according to the grouping. After 24 hours of incubation, CCK-8 levels were measured. Figure 2 As shown in (B), the CCK-8 results showed that Dex treatment for 24 h significantly reduced cell proliferation. However, BMSC-CM prepared by different intensities of mechanical stimulation of BMSCs had different inhibitory effects on rescuing MC3T3-E1 cells under Dex treatment. BMSC-CM with mechanical stimulation intensities of 5%, 10%, and 15% could all rescue MC3T3-E1 cells under Dex treatment, but 10% mechanical stimulation had the best effect on cell proliferation.

[0060] EdU cell proliferation assay: MC3T3-E1 cells were seeded in 24-well plates and treated in groups for 24 hours. EdU working solution was added and incubated for 2 hours. Cells were fixed, permeabilized, and incubated with Click reaction in the dark for 30 minutes. Nuclei were stained with DAPI, and EdU cells were counted using a fluorescence microscope. + Cells (green) / DAPI + Cells (blue), such as Figure 2As shown in (CD), the EDU experiment yielded similar results to the CCK-8 experiment;

[0061] RT-PCR osteogenic gene detection: such as Figure 2 As shown in (EF), compared with the Control group, the relative expression level of osteogenic markers (RUNX2, OPN) mRNA was significantly decreased in the Dex group. The relative expression levels of the Dex+5%, Dex+10%, and Dex+15% groups were higher than those in the Dex group, and the relative expression level of the Dex+10% group was higher than that of the other groups.

[0062] Example 4:

[0063] Exosomes (Exo) and mechanically stimulated exosomes (MS-Exo) were extracted from the culture supernatant of BMSCs and characterized by TEM, NTA and WB.

[0064] Transmission electron microscopy (TEM) observation: Exosome suspension was dropped onto a copper grid, negatively stained with 2% phosphotungstic acid for 1 minute, dried at room temperature, and then the vesicle morphology was observed under TEM. Figure 3 As shown in (A), BMSC-Exo has a vesicle-like structure with a size of approximately 150 nm.

[0065] Nanoparticle tracking analysis (NTA): Exosome suspension was diluted to 10 μL with PBS. 7 -10 9 particles / mL, particle size distribution detected by NTA instrument, such as Figure 3 As shown in (B), the particle size is mainly concentrated between 100 and 200 nm;

[0066] Western blot biomarker detection: such as Figure 3 As shown in (C), BMSC-Exo surface markers were positive for TSG101 and negative for Calnexin.

[0067] Exosome cell uptake verification: such as Figure 3 As shown in (D), PKH67-labeled BMSC-Exo was successfully taken up by MC3T3-E1, and the exosomes secreted by BMSCs under mechanical stimulation (MS-Exo) had an effect on the proliferation of MC3T3-E1 under Dex treatment.

[0068] The rescue effect of exosomes on Dex inhibition was investigated, and the group design is shown in the table below:

[0069] Group Treatment method (24-hour intervention) Control normal culture medium Dex 50μMDex Dex+Exo 50μM DEx + 50μg / mL Exo Dex+MS-Exo 50 μM DEx + 50 μg / mL MS-Exo

[0070] CCK-8 proliferation assay: MC3T3-E1 cells were seeded in 96-well plates. After grouping and treatment, the proliferation rate was measured at 24h and 48h according to the standard CCK-8 protocol. Figure 3As shown in (E), Exo and MS-Exo rescued the inhibition of MC3T3-E1 proliferation by Dex. MS-Exo had a stronger proliferation effect than Exo, but it was not statistically significant at 48h. Therefore, exosomes play a role within 24h in rescuing the inhibition of MC3T3-E1 proliferation by Dex.

[0071] EdU proliferation assay: Cells were seeded in 24-well plates, treated with different groups for 24 hours, and then incubated with EdU reagent for 2 hours. After fixation, permeabilization, and the Click reaction, EdU was counted under a fluorescence microscope. + / DAPI + Cell ratio, such as Figure 3 As shown in (FG), the EDU experiment yielded similar results to the CCK-8 experiment;

[0072] As can be seen from the above, MS-Exo can better promote the proliferation of MC3T3-E1 under Dex treatment.

[0073] Example 5:

[0074] To verify the promoting effect of MS-Exo on osteogenic differentiation and mineralization of MC3T3-E1 under Dex-inhibition environment;

[0075] The grouping design is shown in the table below:

[0076] Group Treatment approach (full intervention) Control Normal osteogenic induction medium (OM) Dex OM+50μMDex Dex+Exo OM + 50 μM Dex + 50 μg / mL Exo Dex+MS-Exo OM + 50 μM Dex + 50 μg / mL MS-Exo

[0077] Cell seeding and induction: MC3T3-E1 cells were seeded in 6-well plates. When the cell confluence reached 70-80%, the medium was replaced with the osteogenic induction medium corresponding to the group. The medium was changed every 3 days and the induction was continued for 7 days (ALP detection) or 21 days (ARS detection).

[0078] Alkaline phosphatase staining (ALP): On day 7, discard the culture medium, wash twice with PBS, fix with 4% paraformaldehyde for 10 minutes, incubate in BCIP / NBT solution in the dark for 30 minutes (37℃), terminate the reaction with distilled water, take pictures under a microscope, and analyze the staining area ratio using ImageJ software. Figure 4 As shown in (AB), based on the ALP results, the Dex group showed the smallest staining area, while the Dex+Exo-MS group showed a significantly larger staining area compared to the Dex group and the Dex+Exo group.

[0079] Alizarin Red S (ARS) staining of mineralized nodules: On day 21, discard the culture medium, wash twice with PBS, fix with 70% ethanol for 15 minutes, add 2% ARS for staining for 10 minutes, wash with distilled water until the background is colorless, photograph under a microscope, dissolve the stain with 10% hexadecylpyridine chloride, and measure the OD value at 562 nm. Figure 4As shown in (CD), ARS found the same results as ALP;

[0080] osteogenic gene expression qPCR analysis: such as Figure 4 As shown in (EI), the RT-PCR results showed that compared with the Dex group, the mRNA expression levels of (RUNX2, OPN, ALP, OCN, BMP2) in the Dex+Exo and Dex+MS-Exo groups were significantly increased. Among them, the Dex+MS-EXO group showed a higher expression level than the Dex+Exo group. MS-Exo had a greater effect on early and mid-stage osteogenic markers (RUNX2, ALP, OPN, BMP2), but its effect on the late-stage osteogenic marker OCN was not particularly significant.

[0081] As can be seen from the above, MS-Exo can promote osteogenic differentiation and mineralization of MC3T3-E1 in the Dex-treated environment.

[0082] Example 6:

[0083] Principal component analysis (PCA) was performed to assess within-group repeatability and between-group variability.

[0084] Gene expression profiles of three MC3T3-E1 cell groups (Control, Dex, and Dex+MS-Exo) were obtained, and the grouping design is shown in the table below:

[0085] Group Treatment method (24-hour intervention) Biological repetition Group C normal culture medium n＝3 Group D 50μMDex n＝3 Group M 50 μM DEx + 50 μg / mL MS-Exo n＝3

[0086] Bioinformatics analysis: such as Figure 5 As shown in (A), Dim1 accounted for 36.1% and Dim2 accounted for 13.7%. Furthermore, the samples were largely clustered within each group, indicating good sample reproducibility, while the dispersion of the groups indicated significant inter-group differences.

[0087] like Figure 5 (B) Based on the criteria of Fold Change ≥ 1.5, p-value ≤ 0.05, and padj ≤ 1, a total of 3973 differentially expressed genes were identified between groups D and C, with 1906 upregulated and 2067 downregulated in the Dex group;

[0088] like Figure 5 As shown in (C), 733 genes with different expression were identified between groups M and D, with 237 upregulated and 496 downregulated in group M.

[0089] like Figure 5 As shown in (D), the intersection of the differentially expressed genes from Dex vs Control and MS-Exo vs Dex yielded 393 common genes;

[0090] like Figure 5 As shown in (E), the heatmap using the above gene mean values ​​demonstrates the reversal of abnormal MC3T3-E1 gene expression after MS-Exo intervention;

[0091] Differentially expressed genes were used for GO annotation, including biological processes (BP), cellular components (CC), and molecular functions (MF), such as Figure 6 (AB) lists the top 10 most significant items for each item, and these parameters are mainly related to cell division, extracellular matrix structure, extracellular space, same protein binding, and ATP binding.

[0092] like Figure 6 As shown in (C–D), the pathways analyzed by KEGG were Dex vs Control and MS-Exo vs Dex, respectively. KEGG analysis revealed osteogenic signaling pathways, including Wnt, JAK-STAT, TGF-beta, and signaling pathways regulating stem cell pluripotency, with the Wnt pathway being the most significantly enriched. By screening for differentially expressed genes shared by Dex vs Control and MS-Exo vs Dex in the Wnt pathway, it was found that TCF7 was upregulated in MS-Exo vs Dex but downregulated in Dex vs Control.

[0093] like Figure 7 As shown in (EF), TCF7 is located on the lower right side of the Wnt pathway in Dex vs Control, while this is exactly the opposite in MS-Exo vs Dex;

[0094] As shown above, this indicates that TCF7 in the Wnt pathway is an important influencing factor under Dex treatment, and can be significantly upregulated by MS-Exo.

[0095] Example 7:

[0096] The Wnt / β-catenin pathway mechanism was validated, and the grouping design is shown in the table below:

[0097] Group Treatment method (24-hour intervention) Control normal culture medium Dex 50μMDex Dex+MS-Exo 50 μM DEx + 50 μg / mL MS-Exo Dex+MS-Exo+Dkk-1 50μDex+50μg / mLMS-Exo+10μg / mLDkk-1

[0098] Immunofluorescence detection of β-catenin nuclear translocation:

[0099] like Figure 7 As shown in (A), the fluorescence intensity in the Dex group was significantly lower than that in the Control group. The fluorescence intensity in the MS-Exo group increased significantly after intervention, and Dkk-1 weakened the therapeutic effect of MS-Exo. The fluorescence intensity of Dex+MS-Exo+Dkk-1 was significantly lower than that of MS-Exo.

[0100] qPCR detection of Wnt pathway gene expression: such as Figure 7 As shown in (BD), MS-Exo significantly upregulated the relative mRNA expression of β-catenin, LRP6, and TCF7 in RT-PCR experiments.

[0101] like Figure 7 As shown in (E), however, the negative regulator of the Wnt / β-catenin pathway, GSK-3β, was significantly inhibited by MS-Exo;

[0102] As shown above, MS-Exo regulates the Wnt / β-catenin pathway by increasing the expression of LRP6, β-catenin and TCF7 and decreasing the expression of GSK-3β, thereby partially offsetting the effect of Dex. There was no significant difference between the Dex+MS-Exo+Dkk-1 group and the Dex group, indicating that Dkk-1 successfully inhibited the Wnt / β-catenin pathway.

[0103] Verification of osteogenic function after Dkk-1 blockade:

[0104] like Figure 8 As shown in (AC), based on the ALP results, the Dex group showed the smallest staining area, while the Dex+MS-Exo group showed the largest staining area, and Dkk-1 weakened the therapeutic effect of MS-Exo.

[0105] like Figure 8 As shown in (BD), ARS found similar results to ALP;

[0106] Furthermore, MS-Exo treatment increased the relative mRNA levels (RUNX2, OCN, OPN, ALP). However, the addition of Dkk-1 resulted in a decrease in the relative mRNA expression levels (RUNX2, OCN, OPN, ALP) (Figure EH).

[0107] As shown above, MS-Exo regulates osteogenic differentiation of MC3T3-E1 by activating the Wnt / β-catenin signaling pathway, and Dkk-1 counteracts the promoting effect of MS-Exo.

[0108] Example 8:

[0109] like Figure 9 As shown, by establishing a SONFH rat model and intervening with mechanically stimulated functional exosomes, subsequent Micro-CT scans revealed that mechanically stimulated functional exosomes could significantly exert a therapeutic effect on SONFH.

[0110] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing functionalized bone marrow mesenchymal stem cell exosomes via mechanical stimulation, characterized in that, Includes the following steps: (1) Extraction of bone marrow mesenchymal stem cells (BMSCs); (2) Apply periodic tensile mechanical stimulation with an intensity of 10% to BMSCs; (3) Collect the cell supernatant, filter it through 0.22 μm, and extract exosomes by ultracentrifugation to obtain functionalized exosomes MS-Exo.

2. The method for preparing functionalized bone marrow mesenchymal stem cell exosomes by mechanical stimulation according to claim 1, characterized in that: The mechanical stimulus is uniaxial tension with a frequency of 0.5–2 Hz and a duration of 6–48 hours.

3. The method for preparing functionalized bone marrow mesenchymal stem cell exosomes by mechanical stimulation according to claim 1, characterized in that: The BMSCs were identified as having a positive expression rate of CD90 and CD29 ≥95%, a negative expression rate of CD45 ≤2%, and the ability to differentiate into osteoblasts, chondrocytes, and adipocytes.

4. A functionalized bone marrow mesenchymal stem cell exosome, characterized in that: The exosomes are prepared by the method described in any one of claims 1–3, and have a particle size of 100–200 nm, are positive for the surface marker TSG101 and negative for Calnexin.

5. The functionalized bone marrow mesenchymal stem cell exosome according to claim 4, characterized in that: The exosomes contain active ingredients that regulate the Wnt / β-catenin signaling pathway, including upregulation of TCF7, β-catenin, LRP7 expression and / or downregulation of GSK-3β expression.

6. The use of the functionalized exosomes according to claim 4 in the preparation of a drug for treating hormone-induced avascular necrosis of the femoral head.

7. A pharmaceutical preparation for treating hormone-induced avascular necrosis of the femoral head, characterized in that... It comprises: the functionalized exosomes and carrier as described in claim 4; The concentration of the exosomes was 50–200 μg / mL; The carrier is physiological saline or phosphate buffer.

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