Dedifferentiated adipocyte-derived exosome as well as preparation and application thereof

Abstract

The invention relates to the technical field of biomedical materials, in particular to a dedifferentiated adipocyte-derived exosome as well as preparation and application thereof. The method comprises the following steps: incubating dedifferentiated adipocytes, and performing osteogenic induction culture; continuously culturing by using a complete culture medium without exosomes; and finally collecting the supernatant and carrying out separation treatment to obtain the exosome with a disc-shaped double-layer membrane structure and a diameter of 79.5-219.1 nm. The blank of the DFATs source exosome in regenerative medicine in the prior art is filled, and the DFATs source exosome is prepared for the first time and is successfully applied to repair of craniomaxillofacial bone defects.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to exosomes derived from dedifferentiated fat cells and the preparation and application thereof. Background Art

[0002] Craniomaxillofacial bone defects can affect facial aesthetics and even lead to functional impairment. They can be caused by a variety of factors, including infection, tumors, trauma, congenital deformities, and other skeletal disorders. Critical-size bone defects are defined as defects that reach a certain size, often exceeding the natural healing capacity of bone tissue. This condition is clinically complex and challenging to repair, requiring specific strategies and techniques to promote bone regeneration and healing.

[0003] Critical-sized bone defects are primarily treated clinically with two approaches: autologous / allogeneic bone transplantation and biomaterial and scaffold implantation. However, autologous bone transplantation has drawbacks such as significant surgical trauma, prolonged surgery, and poor survival after transplantation; allogeneic transplantation can lead to disease transmission and immune rejection; and scaffold implantation faces challenges such as complex procedures, poor biocompatibility, and the occasional need for a secondary surgery. Therefore, exploring new treatment strategies for critical-sized bone defects remains challenging.

[0004] Stem cell-derived exosomes are becoming a research hotspot in the field of critical bone defect repair. Exosomes are small vesicles secreted by cells that contain a variety of bioactive molecules and play a vital role in intercellular communication. Exosomes regulate repair and regeneration at the site of injury by influencing the proliferation, migration, differentiation, and immune environment of recipient cells. Compared with cell therapy, exosomes have lower immunogenicity, lower mass production costs, better stability, simpler sterilization methods, and more convenient storage conditions. Exosomes can also be applied via local injection or combined with biomaterials, making the procedure relatively simple. These characteristics make exosome-based cell-free therapy a promising alternative strategy to stem cell therapy.

[0005] Currently, there is still a lack of exosomes that are specific for bone tissue repair and can effectively promote new bone formation. DFATs is an excellent seed cell in the field of tissue engineering, but there are no reports on the application of exosomes derived from them in the field of tissue regeneration. Summary of the Invention

[0006] To address the above issues, the present invention aims to provide exosomes derived from dedifferentiated adipocytes, as well as their preparation and application. The dedifferentiated adipocyte-derived exosomes provided by the present invention can promote the osteogenic differentiation of bone marrow mesenchymal stem cells and promote new bone formation in vivo, and can be used to treat craniofacial bone defects and other related diseases.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] The first object of the present invention is to provide exosomes, wherein the exosomes are prepared from dedifferentiated adipocytes, and the exosomes have a disc-shaped double-layer membrane structure with a diameter of 79.5 nm to 219.1 nm.

[0009] A second object of the present invention is to provide a method for preparing exosomes, comprising the following steps:

[0010] (S1) Dedifferentiated adipocytes were incubated and then cultured for osteogenic induction;

[0011] (S2) After step (S1), continue culturing with complete medium without exosomes;

[0012] (S3) After step (S2), the supernatant is collected and separated to obtain exosomes (also known as "conditioned" exosomes).

[0013] In one embodiment of the present invention, in step (S1), during the incubation process, the temperature is 37° C., the time is 2 to 3 weeks, and the culture medium is a high-glucose DMEM medium containing 10 to 20% fetal bovine serum and 1% penicillin-streptomycin.

[0014] In one embodiment of the present invention, in step (S1), during the osteogenic induction culture process, the temperature is 37° C., the time is 3 to 14 days, and the culture medium is an osteogenic induction medium;

[0015] Preferably, the time is 14 days.

[0016] In one embodiment of the present invention, in step (S2), the separation treatment is to place the collected supernatant at 0-4°C for differential gradient centrifugation;

[0017] The differential gradient centrifugation process is as follows:

[0018] The collected supernatant was centrifuged at 300×g, and then the supernatant was centrifuged at 2000×g, and then the supernatant was centrifuged at 1000×g. The supernatant was filtered through a 0.22 μm filter and centrifuged at 110,000×g.

[0019] The third object of the present invention is to provide a use of exosomes in the preparation of a bone defect repair agent.

[0020] The fourth object of the present invention is to provide a bone defect repair agent, comprising a curable carrier and the above-mentioned exosomes;

[0021] Wherein, the exosomes are dispersed in a solidifiable carrier.

[0022] In one embodiment of the present invention, the curable carrier is a photocurable hydrogel;

[0023] The dosage ratio of the photocurable hydrogel to exosomes is 1 mL: 10-50 μg;

[0024] Preferably, the ratio of the photocurable hydrogel to exosomes is 1 mL:10 μg.

[0025] The fifth object of the present invention is to provide a use of a bone defect repair agent in the preparation of a product for treating bone defects.

[0026] The sixth object of the present invention is to provide a product for treating bone defects, wherein the product contains the above-mentioned bone defect repairing agent.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention fills the gap in the existing technology of DFATs-derived exosomes in regenerative medicine. It is the first time to prepare DFATs-derived exosomes and successfully apply them to the repair of critical bone defects in the craniomaxillofacial region.

[0029] (2) DFATs are derived from adipose tissue, which has a large cell reserve and is easy to obtain. Compared with adipose-derived stem cells (ASCs), which are also derived from adipose tissue, DFATs have higher homogeneity, stronger proliferation ability, and higher osteogenic differentiation potential, making them an excellent seed cell that is superior to ASCs.

[0030] (3) Compared with cell therapy, exosomes have lower immunogenicity, lower batch production costs, better stability, simpler sterilization methods, and more convenient storage conditions. These characteristics make cell-free therapy based on DFATs-derived exosomes a promising alternative strategy for cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the morphology of exosomes under a transmission electron microscope;

[0032] Figure 2 This is the result of nanoparticle tracking analysis of exosomes;

[0033] Figure 3 Figure 1 is the immunoblotting result of exosome markers;

[0034] Figure 4 Schematic diagram of alkaline phosphatase staining results of exosomes from different days of osteogenic induction acting on BMSCs at different concentrations;

[0035] Figure 5Schematic diagram of the alkaline phosphatase activity test results of exosomes derived from different days of osteogenic induction acting on BMSCs at different concentrations;

[0036] Figure 6 This is a schematic diagram of the results after alkaline phosphatase staining;

[0037] Figure 7 This is a schematic diagram of the results of Alizarin Red staining;

[0038] Figure 8 Schematic diagram of qRT-PCR results;

[0039] Figure 9 Figure 1 is the immunoblotting result of osteogenesis-related proteins;

[0040] Figure 10 Comparison of micro-CT images after skull defect repair in rats in different treatment groups;

[0041] Figure 11 The following are statistical analysis of indicators such as bone volume fraction, trabecular number, trabecular thickness, and trabecular separation after repair of rat skull defects in different treatment groups;

[0042] Among them, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION

[0043] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0044] In the following examples, unless otherwise specified, all reagents used are commercially available reagents, and all detection means and methods used are conventional detection means and methods in the art.

[0045] Example 1

[0046] This example provides the culture of dedifferentiated adipocytes (DFATs) and the preparation of exosomes therefrom.

[0047] (S1) Adipose tissue was isolated from the bilateral inguinal subcutaneous tissue of 10-week-old female SD rats and minced with ophthalmic scissors. The adipose tissue was then digested with 0.1% (w / v) collagenase (dissolved in high-glucose DMEM) in a 37°C incubator for 1 hour. The digested product was centrifuged at 135 × g for 3 minutes.

[0048] (S2) After step (S1), the digestion product is separated into three layers, namely, from top to bottom, a floating layer containing mature adipocytes, a transparent liquid portion, and a stromal vascular portion at the bottom; high-glucose DMEM medium containing 20% ​​fetal bovine serum and 1% penicillin-streptomycin is prepared, 50 mL of the above medium (high-glucose DMEM medium containing 20% ​​fetal bovine serum and 1% penicillin-streptomycin) is taken and placed into a T25 culture flask, 1 mL of cells from the floating layer is collected and inoculated into the T25 culture flask; the above medium is continued to be taken until the T25 culture flask is completely filled, and the T25 culture flask is then inverted in a 37°C 5% CO2 cell culture incubator;

[0049] (S3) After 7 days of incubation, the T25 culture flask was flipped 180° to obtain adherent DFATs. The culture medium was replaced with high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. After the DFATs density reached 80%, the cells were washed twice with PBS, 1 mL of trypsin was added, and digestion was carried out for 2 minutes. Then, 4 mL of the above-mentioned culture medium (high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin) was added to terminate the digestion. The cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant was discarded, and 2 mL of the above-mentioned culture medium (high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin) was added to resuspend the cells. After pipetting evenly, the cells were inoculated into cell culture dishes (9 mL of high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was pre-added in the culture dishes) at a ratio of 1:2 and transferred to a 37°C 5% CO2 cell culture incubator for culture. When the confluence of the third generation DFATs reached 60%, the high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was replaced with an osteogenic induction medium, and osteogenic induction culture was performed.

[0050] (S4) After 14 days of osteogenic induction, high-glucose DMEM medium containing 10% exosome-free premium fetal bovine serum and 1% penicillin-streptomycin (i.e., complete medium without exosomes) was prepared. The osteogenic induction medium was replaced with complete medium without exosomes. After 48 hours of culture, the cell supernatant was collected into a 50-mL centrifuge tube.

[0051] (S5) The cell supernatant collected in the 50 mL centrifuge tube was centrifuged at 4°C, 300×g for 10 min; the supernatant was placed in a new 50 mL centrifuge tube and centrifuged at 4°C, 2000×g for 10 min; the supernatant was further placed in a new 50 mL centrifuge tube and centrifuged at 4°C, 10,000×g for 30 min, and the supernatant was retained.

[0052] (S6) The supernatant was filtered through a 0.22 μm filter, and the filtered supernatant was added to a centrifuge tube for ultracentrifuge, and centrifuged at 110,000×g for 2 h at 4° C., and the supernatant was discarded.

[0053] (S7) Resuspend the exosome pellet at the bottom of the ultracentrifuge tube with 200 μL phosphate buffer saline (PBS); measure the exosome protein concentration using the BCA assay and record the concentration; store the exosome suspension at -80°C.

[0054] Example 2

[0055] This example provides the identification of exosomes (prepared in Example 1).

[0056] The morphology of exosomes was observed using transmission electron microscopy (e.g. Figure 1 The particle size of exosomes was determined by nanoparticle size analyzer (as shown in Figure 2 ), and the expression of surface markers was detected by Western blotting analysis (as shown in Figure 3 shown).

[0057] pass Figure 1 It can be found that the exosomes obtained have a typical disc-shaped double-layer membrane structure; Figure 2 It can be found that the diameter of exosomes ranges from 79.5nm to 219.1nm, with a peak size of approximately 118.8nm. Figure 3 It can be found that exosomes express two positive markers: CD81 and TSG101; the endoplasmic reticulum membrane marker Calnexin is negative in exosomes, but positive in DFATs lysate (marked as "cell lysate" in the figure).

[0058] Example 3

[0059] To determine the optimal days and concentrations of DFATs-derived exosomes for osteogenic induction, exosomes were isolated from the supernatant after 0, 3, 7, and 14 days of osteogenic induction and named 0d-Exo, 3d-Exo, 7d-Exo, and 14d-Exo, respectively. To determine the osteogenic effects of DFATs-derived exosomes on bone marrow-derived mesenchymal stem cells (BMSCs), BMSCs were exposed to different groups of exosomes. After 7 days, ALP staining was performed using a BCIP / NBT alkaline phosphatase colorimetric kit, and ALP activity was measured using an alkaline phosphatase detection kit.

[0060] BCIP / NBT alkaline phosphatase (ALP) color development ( Figure 4) The results showed that DFATs-derived exosomes with different exosome concentrations (10, 25, 50 μg / mL) and different osteogenic induction days (0, 3, 7, 14 days) had different abilities to promote BMSCs osteogenesis. Combined with ALP activity quantitative detection ( Figure 5 ) showed that when exosomes from 14 days of osteogenic induction were applied to BMSCs at a concentration of 10 μg / mL, the ALP activity in this group was significantly higher than in other groups. Therefore, a concentration of 10 μg / mL was selected for subsequent experiments, using 14 days of osteogenic induction as the "conditioning" pretreatment for exosomes.

[0061] Example 4

[0062] This example provides the osteogenic differentiation effect of DFATs-derived exosomes (prepared in Example 1) on BMSCs.

[0063] BMSCs were seeded into cell culture plates and cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. When the cells reached 60% confluence, the medium was replaced with osteogenic induction medium for osteogenic induction culture.

[0064] According to the content of exosomes in the osteogenic induction medium, the mice were divided into two groups: a control group (osteogenic differentiation induction medium + PBS) and an experimental group (osteogenic differentiation induction medium + 10 μg / mL exosomes). The volume of PBS added to the control group was the same as that of the experimental group.

[0065] 1) Alkaline phosphatase staining

[0066] After 7 days of osteogenic induction, alkaline phosphatase staining was performed using the BCIP / NBT alkaline phosphatase colorimetric kit (Beyotime, China) (results are shown in Figure 6 shown).

[0067] The results showed that exosomes can promote the expression of ALP in BMSCs, that is, they can promote the osteogenic differentiation of BMSCs.

[0068] 2) Alizarin red staining

[0069] After 21 days of osteogenic induction, 2 mL of 4% neutral formaldehyde solution (Biosharp, China) was taken for fixation at room temperature for 30 minutes, and alizarin red dye was added for staining. The staining effect was observed under a microscope and images were collected (the results are shown in the figure). Figure 7 shown).

[0070] The results showed that the number of mineralized nodules formed in the exosome group was greater than that in the control group, indicating that exosomes can promote the osteogenic differentiation of BMSCs.

[0071] 3) Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR)

[0072] Fresh osteogenic induction medium was replaced every 3 days. After 7 days of osteogenic induction, total RNA was extracted from the experimental and control groups using Trizol reagent (Takara, Japan). The RNA was reverse transcribed into cDNA using PrimeScrip™ RT kit (Takara, Japan).

[0073] Quantitative polymerase chain reaction was performed using SYBR reagent (YEASEN, China) with GAPDH as the internal reference; the mRNA expression of osteogenesis-related genes was detected using 2 -ΔΔCt Quantitative analysis was performed (the results were as follows Figure 8 shown).

[0074] pass Figure 8 It was found that exosomes could significantly upregulate the expression levels of osteogenesis-related genes BMP2 (p=0.0025), RUNX2 (p=0.0002), OCN (p=0.0013), and OPN (p<0.0001).

[0075] 4) Western blotting to detect the expression of osteoblast-related proteins

[0076] After 7 days of osteogenic induction, total protein was extracted using RIPA lysis buffer (Merck, USA); after protein denaturation, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed.

[0077] The proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane; the membrane was blocked with blocking solution (Beyotime, China); incubated with primary antibody at 4°C overnight; then incubated with secondary antibody (YEASEN, China) for 1 h; and developed with enhanced chemiluminescence reagent (Thermo Fisher Scientific, USA) (the results are shown in Figure 2). Figure 9 shown).

[0078] The results showed that exosomes could significantly promote the expression of osteoblast-related proteins BMP2 and RUNX2 in BMSCs.

[0079] Example 5

[0080] This example provides the effect of DFATs-derived exosomes (prepared in Example 1) on rat bone regeneration.

[0081] The exosomes were fused and mixed with 10 wt% of methacryloyl gelatin (GelMA) to a final exosome concentration of 10 μg / mL; an exosome-hydrogel complex was successfully constructed as a bone damage repair agent.

[0082] Under copious saline irrigation, a trephine was used to drill holes at low speed to construct critical skull defects with a diameter of 5 mm on both sides of the midline of the rat skull. The rats were divided into three groups according to the treatment of the defect area: 1) untreated group; 2) hydrogel group (only methacryloylated gelatin was used); 3) hydrogel + exosome group (exosome-hydrogel complex).

[0083] During the operation, the bone repair agent was injected into the defect site according to the group, and the 365nm 60mW / cm 2 Irradiate under ultraviolet light source for 30 seconds to transform the bone damage repair agent from liquid phase to solid phase.

[0084] The wound was sutured in layers according to the periosteum, subcutaneous tissue and skin, and penicillin was injected intramuscularly after surgery to prevent infection.

[0085] The rats were killed 8 weeks after surgery, and the bone volume fraction, trabecular thickness, number, and separation were detected using micro-CT (the results are shown in Figure 10 and Figure 11 shown).

[0086] pass Figure 10 and Figure 11 It can be found that micro-CT results show that hydrogel loaded with DFTAs-derived exosomes can promote the repair of rat skull defects. The bone volume fraction (0.2396±0.03708%) and the number of trabeculae (0.8706±0.1121mm) of the hydrogel + exosome group were significantly improved. -1 ), trabecular thickness (0.4727±0.1041mm) and trabecular separation (0.8083±0.09479mm) all showed better bone repair effects, and were statistically significant.

[0087] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the explanations of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. An exosome, characterized in that: The exosomes are prepared from dedifferentiated adipocytes, and have a disc-shaped double-layer membrane structure with a diameter of 79.5 nm to 219.1 nm.

2. A method for preparing exosomes, characterized in that: The following steps are involved: (S1) Dedifferentiated adipocytes were incubated and then cultured for osteogenic induction; (S2) After step (S1), continue culturing with complete medium without exosomes; (S3) After step (S2) is completed, the supernatant is collected and separated to obtain exosomes.

3. The method for preparing exosomes according to claim 2, characterized in that: In step (S1), during the incubation process, the temperature is 37° C., the time is 2 to 3 weeks, and the culture medium is a high-glucose DMEM culture medium containing 10 to 20% fetal bovine serum and 1% penicillin-streptomycin.

4. The method for preparing exosomes according to claim 2, characterized in that: In step (S1), during the osteogenic induction culture process, the temperature is 37°C, the time is 3 to 14 days, and the culture medium is osteogenic induction culture medium.

5. The method for preparing exosomes according to claim 2, characterized in that: In step (S2), the separation treatment is to place the collected supernatant at 0-4°C for differential gradient centrifugation; The differential gradient centrifugation process is as follows: The collected supernatant was centrifuged at 300×g, and then the supernatant was centrifuged at 2000×g, and then the supernatant was centrifuged at 10000×g. The supernatant was filtered through a 0.22 μm filter and centrifuged at 110000×g after filtration.

6. Use of the exosomes according to claim 1 in preparing a bone defect repairing agent.

7. A bone defect repairing agent, characterized in that: The bone defect repair agent comprises a curable carrier and the exosomes according to claim 1; Wherein, the exosomes are dispersed in a solidifiable carrier.

8. The bone defect repairing agent according to claim 7, characterized in that: The curable carrier is a photocurable hydrogel; The dosage ratio of the photocurable hydrogel to the exosomes is 1 mL: 10-50 μg.

9. Use of the bone defect repairing agent according to any one of claims 7 to 8 in the preparation of a drug for treating bone defects.

10. A product for treating bone defects, characterized in that: The product contains the bone defect repairing agent according to any one of claims 7 to 8.

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