Application of overexpressed AQP1 mitochondria in preparation of products for treating bone defects

Abstract

The invention discloses application of overexpressed AQP1 mitochondria in preparation of products for treating bone defect, stem cells of overexpressed AQP1 are constructed through a lentiviral transfection method, overexpressed AQP1 mitochondria is obtained, and the obtained mitochondria has mitochondrial structure and function integrity and is enriched with AQP1 protein; in an in-vitro experiment, the overexpressed AQP1 mitochondria can inhibit the polarization and osteoclast differentiation capability of macrophages and relieve inflammation; a periodontitis animal model research shows that overexpression AQP1 mitochondrial transplantation can regulate and control a bone immune microenvironment and relieve tissue inflammation damage; the bone resorption can be inhibited, the damage of tissue collagen fibers is reduced, and the bone defect repair and regeneration of diseases such as periodontitis are promoted. Therefore, the overexpressed AQP1 mitochondria can be used for precise treatment of diseases such as periodontal tissue or bone tissue defects, the treatment effect is remarkably superior to that of common mitochondria, and the overexpressed AQP1 mitochondria has good transformation application prospects in the fields of tissue engineering and cell-free treatment.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly, to the application of overexpressed AQP1 mitochondria in the preparation of products for treating bone defects. Background Art

[0002] Bone tissue loss caused by various reasons such as trauma, tumors, bone infections, etc. is called bone defect. Severe bone defects require a long time to recover, and may even lead to permanent loss of the patient's physical function. This seriously affects the quality of life of the patient. At the same time, with the advent of an aging society, the incidence of bone defect diseases has also increased year by year, bringing a heavy burden to the medical system.

[0003] Traditional treatment methods for bone defects mainly include autologous bone transplantation, allogeneic bone transplantation, artificial bone substitute filling, etc. However, the above-mentioned therapies still have many limitations. For example, autologous bone transplantation has additional trauma to the donor site, and the size of the donor is limited; the potential biosafety risks of allogeneic bone transplantation have raised concerns; the unstable degradability of artificial bone substitutes limits their further clinical use. Therefore, it is urgent to develop effective alternative strategies to promote the repair of bone defects, which is of great significance for improving the prognosis of bone defect patients.

[0004] Mitochondrial transplantation is to implant mitochondria extracted from healthy tissues or cells into areas where mitochondria are damaged or missing, in order to restore the function of damaged cells and promote organ repair. This technology has shown its potential in the treatment of many diseases such as neurodegenerative diseases, cardiovascular diseases, immune diseases, etc. Patent CN 113633662A discloses that mitochondria can be captured by human periodontal ligament stem cells damaged by inflammation, enhancing the osteogenic differentiation ability of human periodontal ligament stem cells, regulating the immunomodulatory function of periodontal ligament stem cells in the inflammatory microenvironment, promoting the differentiation of macrophages into M2 type, reducing inflammation, and can be used for the treatment of inflammatory diseases such as periodontitis; studies on animal models of periodontitis show that mitochondrial transplantation can reduce the damage of inflamed periodontal tissues, promote osteogenesis of periodontal tissues, and solve the dilemma that the existing treatment methods cannot effectively restore the lost periodontal tissues. However, the therapeutic effect of mitochondria in reducing the damage of inflamed periodontal tissues and promoting osteogenesis of periodontal tissues is limited. Therefore, it is of great significance to provide a more effective method for reducing tissue inflammatory damage and treating periodontal tissue or bone tissue defects. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies existing in the prior art, and provide an application of overexpressed AQP1 mitochondria in the preparation of products for treating bone defects.

[0006] The second purpose of the present invention is to provide an application of overexpressed AQP1 mitochondria in the preparation of products for promoting the recovery of inflammatory tissues.

[0007] The above object of the present invention is achieved by the following technical solutions:

[0008] AQP1 is an important component of the transmembrane water channel family proteins and plays an important role in regulating functions such as cell senescence, cell migration, and cell proliferation. Research shows that knocking down AQP1 promotes the progression of inflammatory diseases by increasing the secretion of inflammatory mediators and altering the classical activation pathway of macrophages, but the effect of AQP1 in bone defects has not been reported. Therefore, the present invention first constructs the pMSCV-AQP1 plasmid, then obtains the pMSCV-AQP1 lentiviral supernatant by plasmid transfection, and uses the lentiviral transfection method to transfect the constructed pMSCV-AQP1 lentivirus into human mesenchymal stem cells, and screens out the stably transfected AQP1-overexpressing cell line by drugs. Further, mitochondria are extracted from the AQP1-overexpressing cell line by a kit and differential centrifugation method. The obtained mitochondria have the integrity of mitochondrial structure and function and highly express AQP1. In in vitro experiments, the AQP1-overexpressing mitochondria can inhibit the polarization and osteoclast differentiation ability of macrophages, and reduce inflammation; studies on animal models of periodontitis show that transplantation of AQP1-overexpressing mitochondria can regulate the bone immune microenvironment, reduce tissue inflammatory damage; it can also inhibit bone resorption, reduce the destruction of tissue collagen fibers, and promote the repair and regeneration of bone defects in diseases such as periodontitis. Therefore, it can be used for the precise treatment of diseases such as periodontal tissue or bone tissue defects, and the treatment effect is significantly better than that of ordinary mitochondria.

[0009] Therefore, the present invention provides the application of AQP1-overexpressing mitochondria in the preparation of products for treating bone defects.

[0010] Further, the bone defect is a bone defect caused by periodontitis.

[0011] Further, the product repairs and regenerates the bone defect by inhibiting bone resorption, reducing the destruction of tissue collagen fibers or promoting new bone formation.

[0012] The present invention also provides the application of AQP1-overexpressing mitochondria in the preparation of products for promoting the recovery of inflammatory tissues.

[0013] Further, the product promotes the recovery of inflammatory tissues to normal tissues by inhibiting the polarization and osteoclast differentiation of macrophages.

[0014] Further, the inflammatory tissue includes but is not limited to periodontal tissue.

[0015] Further, the preparation method of the AQP1-overexpressing mitochondria is to first construct an AQP1-overexpressing plasmid, then transfect stem cells to obtain AQP1-overexpressing stem cells, and finally extract and isolate the AQP1-overexpressing mitochondria.

[0016] Furthermore, the stem cells are human mesenchymal stem cells; the stem cells can select appropriate separated tissues or cells according to different indications.

[0017] Preferably, when used for treating periodontitis, the mitochondria are isolated from healthy human periodontal ligament stem cells (hPDLSCs).

[0018] Preferably, the transfection is the lentiviral transfection method.

[0019] Preferably, the extraction and separation is to first break the cells to release mitochondria and other organelles, and then separate the mitochondria by differential centrifugation.

[0020] Preferably, the plasmid overexpressing AQP1 is pMSCV-AQP1.

[0021] Furthermore, the concentration of mitochondria overexpressing AQP1 in the product is 0.3 - 0.5 μg of mitochondria per 20 μL.

[0022] Preferably, the concentration of mitochondria overexpressing AQP1 in the product is 0.4 μg of mitochondria per 20 μL.

[0023] Furthermore, the product is a drug.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention provides the application of a mitochondrion preparation overexpressing AQP1 in the preparation of a product for treating bone defects. The present invention constructs stem cell-derived mitochondria overexpressing AQP1 by the lentiviral transfection method. The obtained mitochondria have the integrity of mitochondrial structure and function, and are enriched with AQP1 protein. In vitro experiments show that the mitochondria overexpressing AQP1 can inhibit the polarization and osteoclastic differentiation ability of macrophages, and reduce inflammation. The study on the periodontitis animal model shows that the transplantation of mitochondria overexpressing AQP1 can regulate the bone immune microenvironment and reduce tissue inflammatory damage. It can inhibit bone resorption, reduce the destruction of tissue collagen fibers, and promote the repair and regeneration of bone defects in diseases such as periodontitis. It can be used for the precise treatment of diseases such as periodontal tissue or bone tissue defects, and the treatment effect is significantly better than that of ordinary mitochondria. It has good translational application prospects in the fields of tissue engineering and cell-free therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the transmission electron microscopy result of the mitochondria overexpressing AQP1 extracted and separated.

[0027] Figure 2 It is the particle size distribution result of the mitochondria overexpressing AQP1 extracted and separated.

[0028] Figure 3To extract the results of labeling of mitochondria overexpressing AQP1 separated by the membrane potential dye Mito-Tracker Red CMXRos.

[0029] Figure 4 To detect the AQP1 expression level of mitochondria overexpressing AQP1.

[0030] Figure 5 To investigate the effect of mitochondria overexpressing AQP1 on the osteoclast differentiation ability of macrophages.

[0031] Figure 6 To investigate the effect of mitochondria overexpressing AQP1 on macrophage polarization.

[0032] Figure 7 To investigate the therapeutic effect of mitochondria overexpressing AQP1 on murine periodontitis (micro-CT detection results).

[0033] Figure 8 To investigate the therapeutic effect of mitochondria overexpressing AQP1 on murine periodontitis. Among them, Figure 8 A shows the results of H&E staining; B shows the results of Masson staining. Specific implementation manners

[0034] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0035] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0036] Example 1 Isolation and identification of mitochondria overexpressing AQP1

[0037] I. Experimental methods

[0038] 1. Construction and isolation of mitochondria overexpressing AQP1

[0039] Extract the mitochondria of hPDLSCs (human periodontal ligament stem cells) overexpressing AQP1 for subsequent experiments. The specific method is as follows:

[0040] (1) Design primers according to the target gene sequence of Gene Bank (NM_001329872.2), construct the pMSCV-AQP1 plasmid, and verify by sequencing.

[0041] (2) Plasmid transfection

[0042] A. Take the clone passed by DNA sequencing for shaking culture, and extract the plasmid according to the instructions of the endotoxin-free plasmid miniprep kit.

[0043] B. Inoculate 293T cells in the logarithmic growth phase into a 150 mm culture dish. When the cell confluence rate reaches 50%-60%, the transfection experiment can be started;

[0044] C. Mix the pMSCV-AQP1 or pMSCV-control plasmid with the PIK plasmid at a ratio of 1:1, and add it to a Polyethylenimine L (PEI) solution (1 mg / mL), and incubate at room temperature for 10 minutes;

[0045] D. Add the mixed solution dropwise to the cell culture dish and gently shake to mix evenly;

[0046] E. After 6-8 hours of transfection, discard the culture medium, add fresh complete medium, and place it in the cell culture incubator to complete the transfection.

[0047] (3) Lentivirus infection

[0048] A. After 48 hours of transfection, centrifuge at 2400×g for 10 minutes at 4°C to remove cell debris;

[0049] B. Collect the virus supernatant;

[0050] C. Take hPDLSCs and inoculate 4 6×10 cells per well into a 6-well plate;

[0051] D. When the cell confluence rate reaches 60-70%, add the virus supernatant and polybrene solution to the culture medium, with a final concentration of 8 μg / mL. After gently shaking the culture plate to mix evenly, centrifuge at 2500 rpm at room temperature for 1 hour, and then place it in the incubator for incubation;

[0052] E. After 6-8 hours, remove the virus mixture and replace it with fresh medium.

[0053] (4) 48 hours after lentivirus infection, digest the cells and passage them. Add 2 μg / mL puromycin to the cell culture medium for drug screening. After 3 days of drug screening, collect the cells for Real-time PCR and Western Blot to detect the expression of AQP1, and obtain AQP1-overexpressing hPDLSCs cells.

[0054] (5) Extract AQP1-overexpressing mitochondria according to the mitochondrial extraction kit instructions

[0055] A. Inoculate AQP1-overexpressing hPDLSCs in the logarithmic growth phase into a 150 mm culture dish. When the cell density reaches 90%, digest and centrifuge routinely, collect the cell pellet, resuspend the cell pellet with 2 mL PBS, and transfer the cell suspension to a new 2 mL EP tube;

[0056] B. After centrifuging at 1200 rpm for 5 min, carefully discard the supernatant, and add 800 μL of mitochondrial extraction reagent A. Place the EP tube on a vortex mixer and vortex at maximum speed for 5 s, then immediately incubate on ice for 2 min (strictly control the time and do not exceed 2 min);

[0057] C. Add 10 μL of mitochondrial extraction reagent B to the cell suspension in the above EP tube and vortex at maximum speed for 10 s;

[0058] D. Incubate on ice for 5 min and vortex at maximum speed for 10 s per minute;

[0059] E. Add 800 μl of mitochondrial extraction reagent C to the EP tube and mix by inverting up and down;

[0060] F. Centrifuge at 700 g, 4 °C for 10 min, and transfer the supernatant to a new 2 mL EP tube;

[0061] G. Centrifuge at 3000 g, 4 °C for 15 min, transfer the supernatant to a new 2 mL EP tube, and resuspend the pellet with 500 μL of mitochondrial extraction reagent C;

[0062] H. Centrifuge at 12000 g, 4 °C for 5 min, discard the supernatant, and the pellet is the overexpressed AQP1 mitochondria, place it on ice;

[0063] I. Resuspend the overexpressed AQP1 mitochondrial pellet with PBS solution for later use.

[0064] 2. Transmission electron microscopy to observe the structural integrity of overexpressed AQP1 mitochondria

[0065] The fixed overexpressed AQP1 mitochondria are dehydrated, infiltrated with resin and embedded to make ultrathin sections, and after double staining, they are scanned and photographed under a transmission electron microscope.

[0066] 3. Zetaview measurement of mitochondrial particle size

[0067] Resuspend the separated overexpressed AQP1 mitochondrial pellet in 200 μL of PBS, and use a 1 mL syringe to inject the suspension into the sample cell of the Zetaview instrument at a constant speed, avoiding air bubbles entering the sample cell, detect the particle size range, and repeat the injection and detection more than 3 times.

[0068] 4. Mito-Tracker Red labeling to identify the activity of overexpressed AQP1 mitochondria

[0069] Add 500 μL of overexpressed AQP1 mitochondria at 20 μg / mL to an EP tube, then add 5 μL (10 μM) of Mito-Tracker Red fluorescent staining working solution and mix well. Incubate in an incubator at 37 °C for 20 min. Then, centrifuge at 3000 g at 4 °C for 15 min. Discard the supernatant, wash twice with PBS, and centrifuge at 12000 g at 4 °C for 15 min. Resuspend the overexpressed AQP1 mitochondria with 500 μL of pre-cooled PBS and transfer to a confocal dish. Observe the fluorescence intensity under a fluorescence microscope to judge the activity degree of the overexpressed AQP1 mitochondria.

[0070] 5. Detection of protein expression of overexpressed AQP1 mitochondria by Western Blot

[0071] Prepare the gel, load the sample, perform electrophoresis, transfer the membrane and block it; incubate the antibody: incubate with the primary antibody: rabbit anti-human AQP1, rabbit anti-human COX IV antibodies; after rinsing, incubate with the corresponding secondary antibody of the same species and image on the machine.

[0072] II. Experimental results

[0073] The results of transmission electron microscopy are as Figure 1 shown. The outer membrane of the extracted overexpressed AQP1 mitochondria is continuous and the structure is complete.

[0074] The results of detecting the particle size of the isolated mitochondria by Zetaview are as Figure 2 shown. The particle size of the extracted overexpressed AQP1 mitochondria is concentrated around 600 nm and 900 nm.

[0075] The results of the activity degree of the overexpressed AQP1 mitochondria are as Figure 3 shown. The extracted overexpressed AQP1 mitochondria can be labeled by Mito-Tracker Red and are active mitochondria.

[0076] The results of Western Blot detection are as Figure 4 shown. Compared with the mitochondria derived from normal hPDLSCs, the overexpressed AQP1 mitochondria highly express AQP1 protein.

[0077] Example 2 Effect of overexpressed AQP1 mitochondria on the osteoclast differentiation ability of macrophages

[0078] I. Experimental methods

[0079] Experimental grouping:

[0080] ① Control (CTR): Culture macrophages routinely and change the medium synchronously with the other groups.

[0081] ② Osteoclast induction group (Rankl exposure): Replace the osteoclast induction medium synchronously with the other groups.

[0082] ③Osteoclast induction + CTR-Mito: Treat with a medium containing mitochondria (CTR-Mito) for 24 h, and then routinely and synchronously replace the osteoclast induction medium.

[0083] ④Osteoclast induction + ovAQP1-Mito: Treat with a medium containing overexpressed AQP1 mitochondria (ovAQP1-Mito) for 24 h, and then routinely and synchronously replace the osteoclast induction medium.

[0084] Mouse bone marrow-derived macrophages (BMDMs) in the logarithmic growth phase were seeded at 5×10 4 cells / well in a 48-well plate and cultured. After induction with M-CSF (25 ng / mL) for 48 h, the cells adhered to the wall and the fresh medium was replaced. According to the above grouping, 20 μg / mL mitochondria were added respectively, and after induction with the osteoclast induction medium (DMEM complete medium containing 25 ng / mL M-csf + 40 ng / mL Rankl) for 12 days, TRAP kit staining was performed.

[0085] II. Experimental results

[0086] The results were as Figure 5 shown. After osteoclast induction, macrophages formed multinucleated cells with positive TRAP staining, indicating that in this experiment, macrophages underwent osteoclast differentiation after osteoclast induction; after treatment with ordinary mitochondria, the number of cells with positive TRAP staining formed was slightly lower than that in the simple induction group; while overexpressed AQP1 mitochondria could significantly reduce the number of cells with positive TRAP staining, suggesting that transplantation of overexpressed AQP1 mitochondria could significantly inhibit the osteoclast differentiation ability of macrophages.

[0087] Example 3 Effect of overexpressed AQP1 mitochondria on the polarization level of macrophages

[0088] I. Experimental methods

[0089] The experimental groups were as follows:

[0090] ①Control group (CTR): Routinely culture macrophages

[0091] ②LPS group: Stimulate with DMEM medium containing P.g-LPS at a final concentration of 1 μg / mL for 24 h.

[0092] ③LPS + CTR-Mito group: After stimulation with DMEM medium containing P.g-LPS at a final concentration of 1 μg / mL for 24 h, replace it with a fresh medium containing ordinary mitochondria (CTR-Mito) and treat for 24 h.

[0093] ④LPS + ovAQP1-Mito group: After stimulating with DMEM medium containing P.g-LPS at a final concentration of 1 μg / mL for 24 h, the medium was replaced with fresh medium containing overexpressed AQP1 mitochondria (ovAQP1-Mito) and treated for 24 h.

[0094] Treat the cells according to the above grouping. At the end time point of the experiment, digest the cells, collect the cells into a flow tube, and wash the cells once with serum-free medium. Then add antibodies and incubate in the dark at room temperature for 30 minutes. Wash the cells three times with PBS and centrifuge at 400 g for 5 minutes, then resuspend in 500 μL of ice-cold PBS. Store the cells in the dark on ice and detect the fluorescence intensity with a flow cytometer.

[0095] II. Experimental results

[0096] The flow cytometry results are as Figure 6 shown. After treating macrophages with LPS, the cells were stimulated by inflammation, and the polarization of M1 macrophages marked by CD86 increased. Transplantation of ordinary mitochondria could reduce the number of cells with M1 polarization, and the reduction effect was more obvious in the treatment group with overexpressed AQP1 mitochondria. The above results indicate that overexpressed AQP1 mitochondria can significantly inhibit the M1 polarization of macrophages in the inflammatory microenvironment and reduce the degree of inflammation in the microenvironment.

[0097] Example 4 Therapeutic effect of overexpressed AQP1 mitochondria on periodontal bone defects

[0098] I. Experimental methods

[0099] Experimental grouping:

[0100] ① Normal mouse group (CTR)

[0101] ② Periodontitis mouse group (PD)

[0102] ③ Periodontitis mouse + ordinary mitochondria group (PD + CTR-mito)

[0103] ④ Periodontitis mouse + overexpressed AQP1 mitochondria group (PD + ovAQP1-mito)

[0104] 1. Establish an animal model of periodontitis

[0105] Use the silk ligature method to induce experimental periodontitis in mice. Under general anesthesia, ligate the bilateral maxillary second molars of the mice with silk thread. Observe whether the silk thread ligature falls off and the periodontal tissue status of the mice every two days. After 2 weeks of ligation, evaluate the bone height from the cementoenamel junction to the alveolar crest by micro-CT, and assess the inflammatory level of the periodontal tissue by H&E and Masson staining.

[0106] 2. Implantation of mitochondria

[0107] According to the grouping treatment, 20 μL of PBS or PBS containing mitochondria (ordinary mitochondria extracted from about 3×10 7 hPDLSCs or mitochondria overexpressing AQP1) was injected subcutaneously into the mesial and distal mucosa on the palatal side of the second molar of mice using a microsyringe. The number of mitochondrial injections was 2 times / week, and samples were taken for detection after 2 weeks. The bone height from the cementoenamel junction to the alveolar crest was evaluated by micro-CT, and the inflammatory level of the periodontal tissue was assessed by H&E and Masson staining.

[0108] 3. Evaluation of treatment effect

[0109] (1) micro-CT detection

[0110] The maxillae of mice in each group were collected and fixed in 4% paraformaldehyde for 24 h. The samples were kept moist with absolute ethanol and placed in a scanning tube for micro-CT scanning. The data was exported and three-dimensional reconstruction was performed using Mimics software.

[0111] (2) H&E and Masson staining

[0112] The maxilla samples of each group were collected, fixed with 4% paraformaldehyde, decalcified with 10% EDTA, dehydrated with gradient ethanol, embedded in paraffin, and paraffin sections were made. The paraffin sections were baked, dewaxed, and hydrated. H&E and Masson staining were performed respectively, and sealed with neutral gum. Observation was carried out under a microscope, and scanning records were made with a digital slide scanner.

[0113] II. Experimental results

[0114] The results of micro-CT detection were as Figure 7 shown. Compared with the healthy mice in the Control group, the periodontitis model was successfully constructed by the silk ligation method, which could effectively induce periodontitis in mice and cause bone resorption of the second molar in mice. After local injection of ordinary mitochondria, bone resorption induced by ligation could be inhibited to a certain extent, while mitochondria overexpressing AQP1 could significantly inhibit periodontal bone resorption. It shows that mitochondria overexpressing AQP1 can significantly promote the repair and regeneration of bone defects in diseases such as periodontitis.

[0115] The staining results were as Figure 8As shown, A shows the results of H&E staining, and B shows the results of Masson staining. The results of H&E staining indicate that after ligature-induced periodontitis in mice, bone resorption appears at the alveolar crest, the height of the alveolar bone decreases, and local inflammatory cell infiltration increases. Compared with the periodontitis and normal mitochondria treatment groups, local injection of overexpressed AQP1 mitochondria significantly reduces bone resorption at the alveolar crest. The results of Masson staining show that after ligature-induced periodontitis in mice, the blue-stained collagen fibers in the periodontal tissue are significantly reduced and sparse, and the destruction of collagen fibers in the periodontal tissue after local injection of mitochondria treatment is reduced, while the collagen fibers in the overexpressed AQP1 mitochondria treatment group are restored more completely. This shows that overexpressed AQP1 mitochondria can improve the periodontal inflammatory microenvironment, reduce tissue inflammatory damage, and promote alveolar bone repair and regeneration.

[0116] In summary, AQP1-expressing mitochondria can significantly inhibit the polarization and osteoclastic differentiation ability of macrophages and reduce inflammation; studies on animal models of periodontitis have shown that transplantation of overexpressed AQP1 mitochondria can regulate the bone immune microenvironment and reduce tissue inflammatory damage; it can inhibit bone resorption, reduce tissue collagen fiber destruction, and promote bone defect repair and regeneration in diseases such as periodontitis, and can be used for the precise treatment of diseases such as periodontal tissue or bone tissue defects, and the treatment effect is significantly better than that of normal mitochondria.

[0117] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Application of AQP1 overexpressing mitochondria in the preparation of products for the treatment of bone defects.

2. Application of AQP1 overexpressing mitochondria in the preparation of products that promote the recovery of inflammatory tissues.

3. The application according to claim 1, characterized in that: The bone defect is a bone defect caused by periodontitis.

4. The use according to claim 1, characterized in that: The product repairs and regenerates bone defects by inhibiting bone absorption, reducing tissue collagen fiber destruction or promoting new bone formation.

5. The application according to claim 2, characterized in that: The product promotes the restoration of inflammatory tissue to normal tissue by inhibiting the polarization and osteoclast differentiation of macrophages.

6. The use according to any one of claims 1 or 2, characterized in that: The method for preparing the AQP1 overexpressing mitochondria comprises first constructing an AQP1 overexpressing plasmid, then transfecting stem cells to obtain AQP1 overexpressing stem cells, and finally extracting and separating the AQP1 overexpressing mitochondria.

7. The use according to claim 6, characterized in that: The stem cells are human mesenchymal stem cells.

8. The use according to claim 6, characterized in that: The AQP1 overexpression plasmid is pMSCV-AQP1.

9. The use according to any one of claims 1 or 2, characterized in that: The concentration of AQP1 overexpressing mitochondria in the product is 0.3-0.5 μg mitochondria per 20 μL.

10. The use according to any one of claims 1 or 2, characterized in that: The product described is a drug.

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