Application and preparation method of mesenchymal stem cell outer vesicles for inhibiting miR-221-3p

Abstract

The invention discloses an application of a bone marrow mesenchymal stem cell outer vesicle for inhibiting miR-221-3p and a preparation method of the bone marrow mesenchymal stem cell outer vesicle. Rat bone marrow mesenchymal stem cell outer vesicles (rBMSCs-EVs) are extracted, rBMSCs-EVs (miR-221-3pin-EVs are used for cells, and antiagomiR-221-3p-EVs are used for animals) for inhibiting miR-221-3p are prepared, and in-vitro osteogenic induction differentiation experiments define that the miR-221-3pin-EVs can remarkably inhibit fluorine-induced osteogenic differentiation enhancement. Meanwhile, the fact that the antagomiR-221-3p-EVs can improve the phenomena of bone mineral density increase, bone trabecula number increase, bone trabecula thickening and the like of a sclerotic bone fluorosis rat model in vivo is further proved, and the antagomiR-221-3p-EVs has important significance on clinical treatment of a patient with the bone fluorosis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application and preparation method of bone marrow mesenchymal stem cell extravesicles that inhibit miR-221-3p. Background Technology

[0002] Fluorine is one of the most widely distributed halogens in nature, existing almost entirely in the form of fluorides. Fluorosis is a chronic, metabolic, systemic skeletal disorder caused by long-term excessive fluorine intake. It can cause persistent resting pain, stiffness, and limited joint movement in bones and joints; severe cases can lead to severe motor impairment and dependence on others for daily living. While the etiology is clear, the pathogenesis remains unclear, causing immense suffering and financial burden on patients and their families. Fluorosis presents with complex and diverse pathologies, classifying into sclerotic, osteoporotic, and osteomalacia types. Sclerotic fluorosis is the predominant type clinically, characterized by abnormal proliferation and activity of osteoblasts. Osteoblasts differentiate from bone marrow mesenchymal stem cells (BMSCs) and are primarily responsible for the synthesis, secretion, and mineralization of bone matrix, making them crucial cells in bone formation and development. Therefore, osteogenic differentiation is closely related to the occurrence and development of fluorosis.

[0003] BMSCs are pluripotent stem cells with self-renewal capacity, multipotent differentiation potential, and paracrine regulatory capacity. Under specific induction conditions, they can differentiate into osteoblasts, adipocytes, chondrocytes, etc. Extracellular vesicles (EVs) are small membrane-bound vesicles released from cells into the external environment, containing abundant nucleic acids, proteins, and lipids. They play important roles in various biological processes such as intercellular communication, substance transport, immune regulation, and tissue repair. miRNAs are also important contents of EVs. miRNAs are a class of endogenous non-coding RNAs with a length of 18-25 nucleotides. They regulate gene expression by binding to target mRNAs and are an important mechanism of post-transcriptional regulation. Previous studies by our research group and literature reports have shown that changes in miRNA expression play an important role in the occurrence and development of fluorosis and osteofluorosis. Currently, some studies have used EVs released by BMSCs and the miRNAs they carry for the intervention and exploration of the pathogenesis of bone metabolic diseases such as fracture repair, osteoarthritis, and osteoporosis. However, it is currently unknown whether BMSCs-EVs and BMSCs-EVs with altered miRNA expression can improve bone damage caused by fluorosis.

[0004] Therefore, the present invention provides a method for preparing extravesicles of bone marrow mesenchymal stem cells that inhibit miR-221-3p and its application in the treatment of bone damage caused by sclerotic fluorosis. Summary of the Invention

[0005] The purpose of this invention is to apply the use of bone marrow mesenchymal stem cell extravesicles that inhibit miR-221-3p in the preparation of a drug for treating bone injury in sclerotic fluorosis.

[0006] Another objective of this invention is to provide a method for preparing extracellular vesicles of bone marrow mesenchymal stem cells that inhibit miR-221-3p.

[0007] The applications described in this invention include the use of rBMSCs-EVs that inhibit miR-221-3p in the preparation of drugs that inhibit fluoride-induced osteogenic progenitor cell differentiation enhancement.

[0008] The applications described in this invention also include the use of rBMSCs-EVs that inhibit miR-221-3p in the preparation of drugs that improve bone mass increase and bone tissue pathological changes in rats with sclerotic fluorosis.

[0009] The drug described in this invention is prepared into a pharmaceutically acceptable dosage form by adding pharmaceutically acceptable excipients using conventional pharmaceutical methods.

[0010] Preferably, the dosage form of the present invention is an injection, tablet, suspension, capsule, granule, etc.

[0011] The bone marrow mesenchymal stem cell extravesicles that inhibit miR-221-3p described in this invention are prepared by the following method: Step 1: Obtaining rat bone marrow mesenchymal stem cells (rBMSCs): Primary cells were extracted from the bone marrow of the rat femur and tibia to obtain rBMSCs, which were then cultured in a cell culture incubator. Step 2: Extract rBMSCs-EVs: Take rBMSCs from passages 3-5 in good growth condition, plate them, and continue culturing for 48 hours the next day in complete medium without EVs, under the same conditions as in step 1. Collect the cell supernatant; centrifuge the cell supernatant at differential speed on a high-speed refrigerated centrifuge; mix the centrifuged cell supernatant with vesicle extraction reagent, shake vigorously, and then incubate overnight at 4°C; centrifuge the overnight precipitate at 10000g for 60 min at 4°C; discard the supernatant to obtain rBMSCs-EVs precipitate, and dilute with PBS to 1 μg / μL; Step 3: Prepare rBMSCs-EVs that inhibit miR-221-3p by electroporation: Add 30 μL of 20 μmol / L miR-221-3pinhibitor or 5 nmol of antagomiR-221-3p to every 100 μL of rBMSCs-EVs, mix well, and incubate on ice for 30 min before transferring to an electroporation cuvette. Place the cuvette into the cavity, ensuring the resistance is between 30-50 Ω, and perform electroporation. Finally, incubate the electroporated rBMSCs-EVs on ice for 30 min to allow for recovery, then centrifuge to remove excess transfection solution to obtain miR-221-3p. in -EVs or antagomiR-221-3p-EVs, after being dispensed, should be stored in a -80℃ refrigerator for later use.

[0012] The culture conditions described in step 1 of this invention are 37°C, 5% CO2, and the culture medium is α-MEM medium containing 10% fetal bovine serum.

[0013] The differential centrifugation step in step 2 of this invention is as follows: centrifuge at 300g for 10 min at 4℃ and discard the precipitate; centrifuge the obtained supernatant at 2000g for 10 min at 4℃ and discard the precipitate; then centrifuge the obtained supernatant at 10000g for 60 min at 4℃ and discard the precipitate.

[0014] In step 2 of this invention, the cell supernatant and vesicle extraction reagent are mixed in a ratio of 4:1.

[0015] The electroporation procedure described in step 3 of this invention is as follows: two perforation pulses, each at 100V for 5msec; and five transfer pulses, each at 20V for 50msec.

[0016] The centrifugation conditions described in step 3 of this invention are: centrifugation at 110,000g for 70 minutes at 4°C.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention inhibits miR-221-3p in bone marrow mesenchymal stem cell extravesicles, which can suppress fluorine-induced osteogenic differentiation. miR-221-3p in Experiments on the regulation of osteogenic differentiation by fluoride-exposed rBMSCs-EVs showed that, compared with the electroporated blank rBMSCs-EVs group, fluoride-exposed rBMSCs-EVs exhibited significantly enhanced osteogenic differentiation due to their high expression of miR-221-3p. in The EVs group significantly inhibited fluorine-induced osteogenic differentiation enhancement, while the electrotransfection inhibitor NC group showed no change, indicating that miR-221-3p is effective in in vitro experiments. in -EVs inhibited fluorine-induced enhanced osteogenic differentiation.

[0018] 2. This invention's inhibition of miR-221-3p extracellular vesicles in bone marrow mesenchymal stem cells can significantly improve bone mass increase and bone tissue pathological changes in rats with fluorosis. Micro-CT scans of rat femurs after antagomiR-221-3p-EVs intervention showed that even after 8 weeks of recovery from fluoride exposure, rats still exhibited increased bone mineral density, increased trabecular bone number, and increased trabecular bone thickness, which were significantly improved after antagomiR-221-3p-EVs intervention. HE staining of rat femur sections showed that the internal morphology of bone tissue in the antagomiR-221-3p-EVs group was improved compared to the natural recovery group and the solvent control group, approaching that of the blank control group. Safranin O and Fast Green staining results indicated that antagomiR-221-3p-EVs can improve fluoride-induced bone mass increase in rats. Attached Figure Description

[0019] Figure 1 Results of rBMSCs-EVs identification (where A and B are transmission electron microscopy results, C is Western blot results, and D is nanoflow cytometry results). Figure 2 Results of osteoblast uptake of rBMSCs-EVs exposed to fluorine. Figure 3 osteogenic differentiation of rBMSCs-EVs and fluoride-treated rBMSCs-EVs after intervention with fluoride-exposed osteogenic progenitor cells and induction of differentiation (where A is the electrophoresis result of osteogenic differentiation marker expression, B is the expression level of COL1A1 in each group, C is the expression level of RUNX2 in each group, D is the result of ALP and ARS staining in each group, and E is the result of ARS staining OD value measurement in each group). Figure 4 qRT-PCR was used to detect the electroporation efficiency of miR-221-3pinhibitor in rBMSCs-EVs; Figure 5 miR-221-3p in -Osteoblast uptake of EVs exposed to fluorine; Figure 6 miR-221-3p in - Osteogenic differentiation of fluoride-exposed osteogenic progenitor cells after intervention with EVs and induction of differentiation (where A is the electrophoresis result of osteogenic differentiation marker expression; B is the expression level of COL1A1 in each group; C is the expression level of RUNX2 in each group; D is the result of ALP and ARS staining in each group; E is the result of ARS staining OD value measurement in each group). Figure 7 Changes in dental fluorosis in rats during rat modeling; Figure 8The levels of bone fluoride, urinary fluoride, and blood fluoride in rats after modeling (where A represents bone fluoride, B represents urinary fluoride, and C represents blood fluoride). Figure 9 Micro-CT scan of rat femurs and analysis of results after modeling (where A is micro-CT image of femur in each group; B is bone mineral density level in each group; C is trabecular separation level in each group; D is number of trabeculae in each group; E is trabecular thickness in each group; F is bone volume fraction in each group; G is bone surface area to tissue volume ratio in each group). Figure 10 HE staining results of the distal femur of rats after modeling; Figure 11 Tracing of rBMSCs-EVs in rats with fluorosis; Figure 12 Tracing of antagomiR-221-3p-EVs in rats with fluorosis; Figure 13 Micro-CT scan of rat femur after intervention with antagomiR-221-3p-EVs and analysis of the results (where A is the micro-CT image of each group; B is the bone mineral density level of each group; C is the trabecular separation level of each group; D is the number of trabeculae in each group; E is the trabecular thickness of each group; F is the bone volume fraction of each group; G is the ratio of bone surface area to tissue volume in each group). Figure 14 HE staining of distal femur in rats after intervention with antagomiR-221-3p-EVs; Figure 15 Safranin O and Fast Green staining of the distal femur of rats after intervention with antagomiR-221-3p-EVs. Detailed Implementation

[0020] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0021] Example 1 miR-221-3p in Preparation method of -EVs or antagomiR-221-3p-EVs (1) Obtaining rBMSCs: Primary cells were extracted from rat femoral and tibial bone marrow to obtain rBMSCs, which were cultured in a cell culture incubator at 37 ℃ and 5% CO2 in α-MEM medium containing 10% fetal bovine serum.

[0022] (2) Extraction of rBMSCs-EVs: Recycled rBMSCs from passages 3-5 in good growth condition were plated and cultured for another 48 hours the following day using EV-free complete medium. Cell supernatant was collected. The supernatant was centrifuged at 300 g for 10 min at 4 °C using a high-speed refrigerated centrifuge, and the precipitate was discarded. The resulting supernatant was then centrifuged at 2000 g for 10 min at 4 °C, and the precipitate was discarded to remove dead cells. The supernatant was then centrifuged at 10000 g for 60 min at 4 °C, and the precipitate was discarded to further remove dead cells and cell debris. Finally, the supernatant and vesicle extraction reagent were mixed at a ratio of 4:1, shaken vigorously, and incubated overnight at 4 °C. The overnight precipitate was centrifuged at 10000 g for 60 min at 4 °C. The supernatant was discarded to obtain the rBMSCs-EVs precipitate, which was diluted to 1 μg / μL with PBS.

[0023] (3) Preparation of rBMSCs-EVs inhibiting miR-221-3p by electrotransfection: Add 30 μL of 20 μmol / L miR-221-3p inhibitor or 5 nmol of antagomiR-221-3p to every 100 μL of rBMSCs-EVs, mix well, and incubate on ice for 30 min before transferring to an electroporation cuvette. Place the cuvette into the cavity and press the "Ω" button; the resistance should be between 30-50 Ω. Then press the start button to execute the electroporation program: two puncture pulses at 100 V for 5 msec each; five transfer pulses at 20 V for 50 msec each. Finally, incubate the electroporated rBMSCs-EVs on ice for 30 min to allow them to recover, then centrifuge at 110,000 g for 70 min at 4 °C to remove excess transfection solution, yielding miR-221-3p. in -EVs or antagomiR-221-3p-EVs, after being dispensed, should be stored in a -80 ℃ refrigerator for later use.

[0024] miR-221-3p prepared in Example 1 in -EVs or antagomiR-221-3p-EVs are used in Examples 2-3 Example 2 miR-221-3p in Application of EVs in inhibiting fluorine-induced enhanced osteoblast differentiation (1) Extraction of fluoride-contaminated rBMSCs-EVs: rBMSCs were treated with 300 μmol / L sodium fluoride (NaF) for 48 h and then fluoride-contaminated rBMSCs-EVs were extracted.

[0025] (2) rBMSCs-EVs were labeled with PKH67 green fluorescent dye.

[0026] (3) After osteoblast progenitor cells were cultured and adhered to the wall on the second day, they were induced to differentiate using osteoblast differentiation induction medium containing NaF. Then, PKH67-labeled rBMSCs-EVs were added for co-incubation, and the uptake of rBMSCs-EVs by fluorine-exposed osteoblasts was observed.

[0027] (4) After osteogenic progenitor cells were cultured and adhered to the wall on the second day, rBMSCs-EVs or fluorine-exposed rBMSCs-EVs were added for incubation. At the same time, osteogenic differentiation was induced by NaF-containing osteogenic differentiation induction solution. The effects of rBMSCs-EVs or fluorine-exposed rBMSCs-EVs on fluorine-exposed osteogenic differentiation were observed.

[0028] (5) Electrotransfect the CY3 red fluorescently labeled miR-221-3p inhibitor into rBMSCs-EVs.

[0029] (6) After osteogenic progenitor cells were cultured and adhered to the culture vessel on the second day, they were induced to differentiate using osteogenic induction medium containing NaF. Then, miR-221-3p carrying CY3 fluorescence was added. in - Co-incubation with EVs to observe the effect of fluoride-exposed osteoblasts on miR-221-3p in -EV intake.

[0030] (7) After osteogenic progenitor cells adhered to the culture vessel on the second day, miR-221-3p was added. in -EVs were incubated, and differentiation was induced using an osteogenic differentiation-inducing solution containing NaF. The miR-221-3p was observed. in The effect of fluoride exposure on osteogenic differentiation.

[0031] Example 3: Application of antagomiR-221-3p-EVs in improving bone mass and bone histopathological changes in rats with sclerotic fluorosis (1) SD rats weighing 110-120 g were selected and administered 15 mg / kg NaF by gavage for 3 months to establish a rat model of fluorosis. The control group rats were administered an equal volume of distilled water by gavage. The rats were allowed free access to food and water daily and were housed in an environment with alternating light and dark conditions for 12 hours. Fluorosis was observed in the teeth, and the levels of fluoride in bones, urine, and blood were measured. Micro-CT scans of the femur were taken and bone tissue pathological sections were prepared for HE staining to confirm the successful establishment of the rat model of fluorosis.

[0032] (2) rBMSCs-EVs were labeled with DIR red fluorescent dye and CY5 red fluorescently labeled antagomiR-221-3p was electrotransfected into rBMSCs-EVs.

[0033] (3) Inject rBMSCs-EVs carrying DIR fluorescence or antagomiR-221-3p-EVs carrying CY5 fluorescence into the knee joint cavity of one side of fluorosis rats, and inject PBS into the other side as a control. Observe the absorption of rBMSCs-EVs or antagomiR-221-3p-EVs in the femur and tibia of fluorosis rats.

[0034] (4) AntagomiR-221-3p-EVs were injected into the bilateral knee joint cavities of rats with fluorosis for intervention, once a week for 8 consecutive weeks.

[0035] (5) After the intervention experiment, rat urine was collected; after euthanizing the rats, blood was collected by cardiac puncture, and the bilateral femurs and tibias of the rats were dissected.

[0036] (6) Micro-CT scans were performed on rat femurs to observe changes in femoral imaging and to conduct bone morphometric analysis.

[0037] (7) The content of bone fluoride, urine fluoride and blood fluoride was determined by fluoride ion selective electrode method.

[0038] (8) Take rat femur to make pathological sections.

[0039] (9) HE staining and Safranin O and Fast Green staining were performed on the pathological sections of rat femurs to observe changes in bone tissue morphology and bone mass.

[0040] To verify the effectiveness of the invention, the inventive team conducted a series of experiments, as follows: I. Experimental Procedure 1. Extraction of rBMSCs-EVs Recycled rBMSCs from passages 3-5 in good growth condition. After plating, replace the medium with EV-free complete medium and continue culturing for 48 h. Collect the cell supernatant. Centrifuge the cell supernatant at 300 g for 10 min at 4 °C using a high-speed refrigerated centrifuge, discard the precipitate, and centrifuge the supernatant at 2000 g for 10 min at 4 °C, discard the precipitate again, and remove dead cells. Centrifuge the supernatant at 10000 g for 60 min at 4 °C, discard the precipitate, and further remove dead cells and cell debris. Mix the cell supernatant with vesicle extraction reagent (Shanghai Yisheng Biotechnology Co., Ltd.) at a ratio of 4:1, shake vigorously, and incubate overnight at 4 °C. Centrifuge the overnight mixture at 10000 g for 60 min at 4 °C, discard the supernatant to obtain the rBMSCs-EVs precipitate, resuspend in PBS, filter through a 0.22 μm filter membrane, aliquot, and store at -80 °C for later use. Extraction of fluoride-contaminated rBMSCs-EVs: After treating rBMSCs with 300 μmol / L NaF for 48 h, fluoride-contaminated rBMSCs-EVs were extracted according to the steps described above.

[0041] 2. Identification of rBMSCs-EVs (1) Observe the morphology and structure of rBMSCs-EVs using transmission electron microscopy (TEM): After fixing rBMSCs-EVs with glutaraldehyde, take 5~10 μL and drop it on the copper grid of the carbon support film. Adsorb at room temperature for about 10 min. Then, add 10 μL of 2% phosphotungstic acid solution to the copper grid. After staining rBMSCs-EVs at room temperature for 2 min, remove the excess staining agent. After drying at room temperature, observe under transmission electron microscopy.

[0042] (2) Detection of particle size and concentration of rBMSCs-EVs by nanoflow cytometry: After diluting the rBMSCs-EVs sample with PBS, add it to the sample cell. According to the instrument prompts, adjust the parameters such as Screen Gain and Camera Level to ensure that the particles are clear in the detection screen. Click "Start Camera" to start the detection and record the Brownian motion of rBMSCs-EVs.

[0043] (3) The expression of surface markers HSP70, TSG101, CD81 and Calnexin of rBMSCs-EVs was detected by Western blot.

[0044] 3. Osteogenic induction differentiation osteoblast progenitor cells were 1×10 5Cells were seeded at a density of 100 cells / well into 12-well plates and cultured in complete medium. After cell adhesion on day 2, the osteogenic induction differentiation medium was replaced, and the medium was changed every 2-3 days. On day 7, ALP staining was performed to observe alkaline phosphatase granules, and on day 14, ARS staining was performed to observe osteogenic mineralization nodules.

[0045] 4. Tracing rBMSCs-EVs within fluoride-exposed osteoblasts rBMSCs-EVs were labeled with PKH67 green fluorescent dye. Cell spreaders were seeded on the bottom of 6-well plates, and fluoride-exposed osteoblast precursor cells were cultured and induced to differentiate for 7 days. Then, PKH67-labeled rBMSCs-EVs were added to the fluoride-exposed osteoblast cells and incubated for 6 hours. The culture medium was removed from the cells, washed twice with PBS, fixed with 4% paraformaldehyde for 30 min, and then stained with DAPI at room temperature for 10 min to label the nuclei. The cells were washed twice with PBS. The cell spreaders were removed, fixed on glass slides, and observed and photographed under a laser confocal microscope.

[0046] 5. Regulation of osteogenic differentiation by fluoride exposure by rBMSCs-EVs and fluoride-exposed rBMSCs-EVs rBMSCs-EVs and fluoride-exposed rBMSCs-EVs were added to fluoride-exposed osteogenic progenitor cells for incubation and osteogenic differentiation was induced. After 7 days, proteins were extracted and Western blot experiments were performed to detect the expression of osteogenic differentiation markers, and ALP staining was performed to observe alkaline phosphatase particles. After 14 days, ARS staining was performed to observe the formation of osteogenic mineralization nodules.

[0047] 6. Preparation of rBMSCs-EVs inhibiting miR-221-3p miR-221-3p inhibitor or antagomiR-221-3p was transfected into rBMSCs-EVs using an electroporation instrument. 30 μL of 20 μmol / L miR-221-3p inhibitor or 5 nmol antagomiR-221-3p was added per 100 μL of rBMSCs-EVs and mixed thoroughly. The mixture was incubated on ice for 30 min. Then, 100 μL of the mixture was transferred to an electroporation cuvette using a pipette tip. The cuvette was then placed in the cavity of the cuvette, and the "Ω" button was pressed. The resistance value should be between 30-50 Ω. The start button was then pressed to execute the electroporation program: two puncture pulses at 100 V for 5 msec each; and five transfer pulses at 20 V for 50 msec each. After electroporation, rBMSCs-EVs were placed on ice for 30 min to retrieval, then centrifuged at 110,000 g for 70 min at 4 °C to remove excess transfection solution. After resuspending in PBS, the cells were aliquoted and stored at -80 °C for later use.

[0048] 7.miR-221-3p in -EVs were traced within fluoride-exposed osteoblasts. The miR-221-3p inhibitor carrying CY3 red fluorescence was transfected into rBMSCs-EVs via electroporation. The electroporated rBMSCs-EVs were then added to fluoride-exposed osteoblasts and incubated for 6 h. The cell response to miR-221-3p was observed. in -EV intake.

[0049] 8.miR-221-3p in Regulation of osteogenic differentiation by fluoride exposure by EVs miR-221-3p in -EVs were added to fluorine-exposed osteogenic progenitor cells for incubation and osteogenic differentiation was induced. After 7 days, proteins were extracted and Western blot experiments were performed to detect the expression of osteogenic differentiation markers, and ALP staining was performed to observe alkaline phosphatase particles. After 14 days, ARS staining was performed to observe the formation of osteogenic mineralization nodules.

[0050] 9. Construction and intervention of a rat model of fluorosis SD rats weighing 110-120 g were randomly divided into two groups: the fluoride-exposed group (NaF group) was administered 15 mg / kg of NaF by gavage daily to establish a rat model of sclerosing fluorosis; the normal control group (Control group) was administered an equal volume of deionized water by gavage daily for 3 months.

[0051] Three months after modeling, the occurrence of fluorosis was determined by observing changes in dental fluorosis, measuring bone fluoride, urinary fluoride and blood fluoride levels, taking micro-CT scans of the femur and preparing bone tissue pathological sections for HE staining.

[0052] After successful modeling, the remaining rats in the NaF group were stopped from gavage and randomly divided into 4 groups: (1) Natural recovery group: free access to food and water until the end of the experiment; (2) Solvent control group: 100 μL of PBS was injected into the knee joint cavity of both lower limbs each time; (3) rBMSCs-EVs intervention group (EVs group): 100 μL of rBMSCs-EVs (1 μg / μL) was injected into the knee joint cavity of both lower limbs each time; (4) rBMSCs-EVs intervention group that inhibits miR-221-3p (antagomiR-221-3p-EVs group): 100 μL of anagomiR-221-3p-EVs (1 μg / μL) was injected into the knee joint cavity of both lower limbs each time. All 4 groups of animals were injected once a week for 8 consecutive weeks. (5) The remaining normal control group rats were used as blank control group, and no treatment was given. They were allowed to eat and drink freely every day until the end of the experiment.

[0053] 10. Tracing of rBMSCs-EVs in the femur and tibia of rats After modeling, rBMSCs-EVs were labeled with DIR red fluorescent dye, and then the labeled rBMSCs-EVs were injected into the left knee joint cavity of fluorosis rats. 100 μL PBS was injected into the right side as a control. The rats were euthanized at 6, 24, 48 and 72 h after injection, and the femur and tibia of both lower limbs were taken for observation and photography using a small animal imaging instrument.

[0054] 11. Tracing of antagomiR-221-3p-EVs in the femur and tibia of rats AntagomiR-221-3p carrying CY5 red fluorescence was electrotransfected into rBMSCs-EVs. The transfected rBMSCs-EVs were then injected into the left knee joint cavity of rats with fluorosis, while 100 μL of PBS was injected into the right side as a control. The rats were euthanized at 6, 24, 48 and 72 h after injection, and the femurs and tibias of both lower limbs were collected for observation and recording using a small animal imaging system.

[0055] 12. Determination of urinary fluoride, blood fluoride, and bone fluoride After modeling, urine was collected, and some rats were euthanized. Blood, bilateral femurs, and tibias were collected. The fluoride content in urine, blood, and bone was detected using a fluoride ion-selective electrode method. First, a series of fluoride ion standards were prepared, their equilibrium potential values ​​were measured, and a standard curve was plotted. Urine, serum, and pretreated femur samples were appropriately diluted, and their potential values ​​were measured. The fluoride content in urine, serum, or femur was then calculated based on the standard curve.

[0056] 13. Micro-CT Detection of Rat Femur After the rat femur was cleaned of muscle, it was placed on a micro-CT scanning table. Scanning parameters were set according to experimental requirements, and the scan was initiated using the Cruiser data acquisition software. Raw images were obtained after the scan was completed. The raw images were then reconstructed using the Recon image reconstruction software. The target region (ROI) was analyzed using the Avatar image display and application software. The same region was selected for analysis for each bone, and the analysis parameters were adjusted to obtain the required parameter values ​​and the data were exported.

[0057] 14. Preparation of pathological sections of rat femur Rat femurs were fixed with 4% paraformaldehyde for 24 h, then immersed in decalcification solution (EDTA-Na2) for about one month. The decalcified femurs were rinsed with running water for 2-3 h, then dehydrated using an alcohol gradient: 70% ethanol → 80% ethanol → 90% ethanol, 1 h each, followed by n-butanol for 4-6 h. After clearing with xylene, xylene I (20 min) → xylene II (15 min) → xylene III (30 min) were added sequentially. The femurs were then immersed in paraffin at 60 °C for 3 h and embedded in paraffin. The embedded blocks were cut into 4 μm thick sections using a paraffin microtome, flattened in a warm water bath, retrieved, and dried.

[0058] 15. HE staining of rat femoral sections Rat femoral sections were dewaxed to water and then sequentially immersed in xylene I (5 min) → xylene II (5 min) → xylene III (5 min) for dewaxing. They were then rehydrated sequentially in anhydrous ethanol I (5 min) → anhydrous ethanol II (5 min) → 95% ethanol (5 min) → 85% ethanol (5 min) → 75% ethanol (5 min) → distilled water (5 min). Hematoxylin staining was added for 10 min, and excess staining was washed away with distilled water. Differentiation was performed with differentiation solution for 40 s, followed by washing twice with tap water. Eosin staining was added for 2 min, and excess staining was discarded. The sections were then rapidly dehydrated and sequentially immersed in 75% ethanol (2-3 s) → 85% ethanol (2-3 s) → 95% ethanol (2-3 s) → anhydrous ethanol I (2-3 s) → anhydrous ethanol II (1 min). The sections were cleared twice with xylene for 1 min each time and mounted with neutral resin.

[0059] 16. Rat femoral bone sections stained with Safranin O and Fast Green After routine dewaxing of rat femoral sections to water, stain with freshly prepared Weigert hematoxylin solution for 5 min, then wash with distilled water. Differentiate with acidic differentiation solution for 3-5 s, then wash with distilled water for 10 min. Stain with Fast Green solution for 5 min. Quickly wash the sections with a weak acid solution for 10-15 s to remove residual Fast Green, then air dry. Stain with safranin solution for 3 min, then wash away excess stain with anhydrous ethanol. Dehydrate with anhydrous ethanol for 1 min, then clear twice with xylene, 1 min each time. Mount with neutral resin.

[0060] II. Experimental Results 1. Extraction and identification of rBMSCs-EVs The results of the identification are as follows Figure 1 rBMSCs-EVs were successfully extracted from rBMSCs supernatant using a kit-based method. TEM analysis revealed that rBMSCs-EVs possessed a typical circular, lipid bilayer structure. Western blot analysis showed positive expression of the specific markers HSP70, TSG101, and CD81, while Calnexin was negative. Nanoflow cytometry analysis showed that the average particle size of rBMSCs-EVs was 76.34 nm, and the concentration was 2.61 × 10⁻⁶. 9 The results are consistent with the characteristics of extracellular vesicles.

[0061] 2. Tracing rBMSCs-EVs within fluoride-exposed osteoblasts After adding rBMSCs-EVs labeled with the green fluorescent dye PKH67 to fluoride-exposed osteoblasts and incubating for 6 h, green fluorescence distributed in the cytoplasm could be observed under a laser confocal microscope, indicating that fluoride-exposed osteoblasts can take up rBMSCs-EVs. The results are as follows: Figure 2 .

[0062] 3. Regulation of osteogenic differentiation by fluoride exposure by rBMSCs-EVs and fluoride-exposed rBMSCs-EVs rBMSCs-EVs and fluoride-exposed rBMSCs-EVs were added to fluoride-exposed osteogenic progenitor cells and incubated for 7 days to induce osteogenic differentiation. Western blotting was used to detect the expression of osteogenic differentiation markers COL1A1 and RUNX2 (results are shown below). Figure 3 A, B, C), combined with ALP and ARS staining results (e.g. Figure 3 (D,E) showed that, compared with the fluoride-treated group, fluoride-treated rBMSCs-EVs could further promote fluoride-induced osteogenic differentiation, while normal rBMSCs-EVs had a significant inhibitory effect on fluoride-induced osteogenic differentiation.

[0063] 4. Preparation of rBMSCs-EVs inhibiting miR-221-3p miR-221-3p inhibitor was transfected into rBMSCs-EVs using electroporation, and the results were then detected by qRT-PCR (e.g., Figure 4 The results showed that, compared to the electroporated blank EVs group, miR-221-3p expression was significantly downregulated in EVs electroporated with miR-221-3p inhibitor, while miR-221-3p expression in EVs electroporated with inhibitor NC was not different from that in the electroporated blank EVs group. This suggests that electroporation can successfully transfect miR-221-3p inhibitor into rBMSCs-EVs.

[0064] 5. miR-221-3p in -EVs were traced within fluoride-exposed osteoblasts. After electrotransfecting rBMSCs-EVs carrying the CY3 red fluorescent miR-221-3p inhibitor into fluoride-exposed osteoblasts and incubating for 6 hours, red fluorescence distributed in the cytoplasm was observed using laser confocal microscopy, indicating that miR-221-3p... in -EVs can be taken up by fluorine-exposed osteoblasts. Results as follows Figure 5 .

[0065] 6. miR-221-3p in Regulation of osteogenic differentiation by fluoride exposure by EVs miR-221-3p in -EVs were added to fluorine-exposed osteogenic progenitor cells and incubated to induce osteogenic differentiation for 7 days. The expression of osteogenic differentiation markers COL1A1 and RUNX2 was detected by Western blotting (results are shown below). Figure 6 A, B, C), combined with ALP and ARS staining results (e.g. Figure 6 (D,E) showed that, compared with the electroporated blank rBMSCs-EVs group, the fluorine-treated rBMSCs-EVs exhibited significantly enhanced osteogenic differentiation due to their high expression of miR-221-3p. in The EVs group significantly inhibited fluoride-induced osteogenic differentiation, while the electroporation inhibitor NC group showed no change. This suggests that miR-221-3p has a significant effect on osteogenic differentiation in vitro. in -EVs inhibited fluorine-induced enhanced osteogenic differentiation, providing a basis for subsequent animal interventions.

[0066] 7. Construction of a rat model of fluorosis Changes in rat teeth with fluorosis during modeling: Figure 7During the 3-month modeling period, the blank control group rats showed no signs of fluorosis. The teeth of the fluoride-treated rats began to show chalky white changes at 1 month of age, with obvious white and brown stripes, which became more pronounced at 3 months. Based on the "Diagnostic Criteria for Fluorosis of the People's Republic of China (WS / T208-2021)" standard, fluorosis can be diagnosed.

[0067] After the model was established, the levels of bone fluoride, urinary fluoride, and blood fluoride in the fluoride-exposed rats were significantly higher than those in the blank control group. (See results below.) Figure 8 micro-CT results are as follows Figure 9 Compared to the blank control group, rats in the fluoride-exposed group showed an increased number of trabeculae, a denser bone structure, and greater bone mass. Bone morphometric analysis revealed that the fluoride-exposed group had higher bone mineral density (BMD), bone volume fraction (BV / TV), and trabeculae number (Tb.N) than the blank control group; however, the trabeculae separation (Tb.Sp) was lower in the fluoride-exposed group than in the blank control group. There were no statistically significant differences in bone surface area to tissue volume ratio (BS / TV) and trabeculae thickness (Tb.Th). These findings suggest that after 3 months of fluoride exposure, rats exhibited increased bone mineral density, bone volume fraction, and trabeculae number, while trabeculae separation decreased.

[0068] HE staining results of the distal femur of rats after modeling are as follows: Figure 10 HE staining results showed that in the blank control group, the trabeculae in the distal femur of rats were regularly arranged, the cortical bone thickness was relatively uniform, and the distribution of hematopoietic cells in the medullary cavity was normal. In the fluoride-treated group, the trabeculae in the distal femur of rats increased and were densely distributed, with reduced separation, thickened cortical bone, and narrowed medullary cavity.

[0069] The above confirms that the rat model of sclerosing fluorosis has been successfully established.

[0070] 8. Tracing of rBMSCs-EVs in the femur and tibia of rats The tracking results are as follows Figure 11 When DIR-labeled rBMSCs-EVs were injected into the left knee joint cavity of rats, the rBMSCs-EVs were mainly concentrated in the knee joint cavity at 6 h after injection, and were then slowly absorbed. By 72 h, they were completely absorbed by the femur and tibia, while no fluorescence was observed in the bones injected with PBS.

[0071] 9. Tracing of antagomiR-221-3p-EVs in the femur and tibia of rats The tracking results are as follows Figure 12 When antagomiR-221-3p-EVs carrying CY5 fluorescence were injected into the left knee joint cavity of rats, rBMSCs-EVs were mainly concentrated in the knee joint cavity at 6 h after injection, and were then slowly absorbed. By 72 h, they were completely absorbed by the femur and tibia, while no fluorescence was observed in the bones injected with PBS.

[0072] 10. Micro-CT scan results of rat femur after intervention with antagomiR-221-3p-EVs micro-CT scan results are as follows Figure 13 Micro-CT imaging of the distal femur in rats showed that the number of trabeculae in the blank control group was normal, the arrangement was neat, the thickness was uniform and the spacing was normal; the number of trabeculae in the natural recovery group and the solvent control group was increased, the arrangement was dense, the structure was disordered, and the spacing between trabeculae was decreased. The antagomiR-221-3p-EVs group showed improvement compared to the natural recovery group and the solvent control group, and was close to the blank control group. The rBMSCs-EVs group also showed some improvement.

[0073] Bone morphometric analysis showed no differences in bone mineral density (BMD), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th), and bone volume fraction (BV / TV) between the natural recovery group and the solvent control group, all of which were higher than those in the blank control group. However, the antagomiR-221-3p-EVs group had lower values ​​than the natural recovery group, and the Tb.N in the antagomiR-221-3p-EVs group was lower than that in the EVs group. There were no differences in trabecular bone separation (Tb.Sp) among the natural recovery group, solvent control group, and EVs group, all of which were lower than those in the blank control group. However, the Tb.Sp in the antagomiR-221-3p-EVs group was higher than that in the EVs group and the natural recovery group. There were no differences in bone surface area and tissue volume ratio (BS / TV) among the five groups. These findings suggest that even after 8 weeks of recovery, rats exposed to fluoride still exhibited increased bone mineral density, increased trabecular bone number, and increased trabecular bone thickness, which were significantly improved after antagomiR-221-3p-EVs intervention.

[0074] 11. HE staining results of rat femoral sections after intervention with antagomiR-221-3p-EVs HE staining results are as follows Figure 14 In the blank control group, the trabeculae in the distal femur of rats were regularly arranged, with relatively uniform thickness and normal spacing. In the natural recovery group and the solvent control group, the trabeculae in the distal femur of rats increased in number and were densely distributed, with decreased separation and narrowing of the medullary cavity. The internal morphology of bone tissue in the antagomiR-221-3p-EVs group was improved compared to the natural recovery group and the solvent control group, and was close to that of the blank control group.

[0075] 12. Results of Safranin O and Fast Green staining of rat femoral sections after intervention with antagomiR-221-3p-EVs Safranin O and Fast Green staining results are as follows Figure 15Clearly visible are orange-red cartilage tissue and blue-green bone tissue. Compared to the blank control group, the bone mass of the distal femur in the natural recovery group and the solvent control group was higher, while the bone mass in the antagomiR-221-3p-EVs group was lower than that in the natural recovery group, approaching that of the blank control group. This suggests that fluoride exposure induces increased bone mass in rats, and antagomiR-221-3p-EVs can play a certain role in improving this effect.

[0076] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An application of a method for inhibiting miR-221-3p in bone marrow mesenchymal stem cell extravesicles, characterized in that, The application is the use of rBMSCs-EVs that inhibit miR-221-3p in the preparation of drugs for treating bone damage in sclerotic fluorosis.

2. The application according to claim 1, characterized in that, The applications include the use of miR-221-3p-inhibiting rBMSCs-EVs in the preparation of drugs that inhibit fluoride-induced osteogenic progenitor differentiation enhancement.

3. The application according to claim 1, characterized in that, The applications also include the use of rBMSCs-EVs that inhibit miR-221-3p in the preparation of drugs that improve bone mass increase and bone tissue pathological changes in rats with sclerosing fluorosis.

4. The application according to claims 1-3, characterized in that, The drug is formulated into a pharmaceutically acceptable dosage form using conventional pharmaceutical methods and pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that, The dosage forms are injections, tablets, suspensions, capsules, and granules.

6. A method for preparing extracellular vesicles of bone marrow mesenchymal stem cells that inhibit miR-221-3p for use in the applications described in claims 1-3, characterized in that, The preparation method includes the following steps: Step 1: Obtain rat bone marrow mesenchymal stem cells (rBMSCs): Primary cells were extracted from rat femoral and tibial bone marrow to obtain rBMSCs, which were then cultured in a cell culture incubator. Step 2: Extract rBMSCs-EVs: Take rBMSCs from passages 3-5 in good growth condition, plate them, and continue culturing for 48 hours the next day using EV-free complete medium, under the same conditions as in step 1. Collect the cell supernatant; centrifuge the cell supernatant at differential speed on a high-speed refrigerated centrifuge; mix the centrifuged cell supernatant with vesicle extraction reagent, shake vigorously, and then incubate overnight at 4 ℃; centrifuge the overnight precipitate at 10000 g for 60 min at 4 ℃; discard the supernatant to obtain rBMSCs-EVs precipitate, and dilute with PBS to 1 μg / μL; Step 3: Prepare rBMSCs-EVs that inhibit miR-221-3p by electroporation: Add 30 μL of 20 μmol / L miR-221-3p inhibitor or 5 nmol of antagomiR-221-3p to every 100 μL of rBMSCs-EVs, mix well, and incubate on ice for 30 min before transferring to an electroporation cuvette. Place the cuvette into the cavity, ensuring the resistance is between 30-50 Ω, and perform electroporation. Finally, incubate the electroporated rBMSCs-EVs on ice for 30 min to allow for recovery, then centrifuge to remove excess transfection solution to obtain miR-221-3p. in -EVs or antagomiR-221-3p-EVs, after being dispensed, should be stored in a -80 ℃ refrigerator for later use.

7. The preparation method according to claim 6, characterized in that, The culture conditions described in step 1 are 37 ℃, 5% CO2, and the culture medium is α-MEM medium containing 10% fetal bovine serum.

8. The preparation method according to claim 6, characterized in that, The differential centrifugation step 2 is as follows: centrifuge at 300 g for 10 min at 4 ℃ and discard the precipitate; centrifuge the obtained supernatant at 2000 g for 10 min at 4 ℃ and discard the precipitate; then centrifuge the obtained supernatant at 10000 g for 60 min at 4 ℃ and discard the precipitate.

9. The preparation method according to claim 6, characterized in that, The cell supernatant and vesicle extraction reagent described in step 2 are mixed in a 4:1 ratio.

10. The preparation method according to claim 6, characterized in that, The electroporation procedure described in step 3 is as follows: two puncture pulses, each at 100 V for 5 msec; five transfer pulses, each at 20 V for 50 msec; and centrifugation conditions: centrifugation at 110,000 g for 70 min at 4 ℃.

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