Preparation method of light-sensitive mesenchymal stem cells and exosomes and application of light-sensitive mesenchymal stem cells and exosomes in promoting osteogenesis

Abstract

The invention discloses preparation methods of light-sensitive mesenchymal stem cells and exosomes and application of the light-sensitive mesenchymal stem cells and the exosomes in promoting osteogenesis, the light-sensitive mesenchymal stem cells have the capability of secreting a large amount of exosomes under near-infrared illumination, are better in safety and can be further used for preparing a bone injury repairing agent, and the bone injury repairing agent can be used for repairing bone injury. And a new choice is provided for pharmaceutical compositions related to bone defect reconstruction.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and specifically to a method for preparing photo-responsive mesenchymal stem cells and exosomes, and their application in promoting osteogenesis. Background Art

[0002] The reconstruction of large craniofacial defects caused by traumatic brain injury is extremely challenging. Autologous transplantation is currently the most effective clinical method for reconstructing large bone defects. However, this method has limitations such as high cost of bone collection, limited bone source, and potential donor site complications. Therefore, autologous / allogeneic bone transplantation is not the best treatment option for bone regeneration. In recent years, a large number of studies have focused on cell therapy. A large number of studies have found that using exogenous mesenchymal stem cells as cell therapy can promote or supplement the repair of endogenous bone defects.

[0003] Mesenchymal stem cells are a type of pluripotent adult stem cells that originate from mesodermal tissues. They are self-renewing pluripotent cells that can secrete a variety of biological factors, creating a regenerative microenvironment with excellent tissue regeneration potential, thereby restoring and repairing injured tissues. Mesenchymal stem cells have a strong ability to express paracrine factors. Exosomes are released by mesenchymal stem cells through a series of physiological processes. Similar to mesenchymal stem cells, exosomes, as a cell-free therapy, have significant immunomodulatory, antioxidant stress, and anti-apoptotic effects, effectively repairing damaged tissues and promoting wound healing.

[0004] "Photo-switch" refers to a rare earth-doped upconversion nanoparticle, especially a lanthanide upconversion material, which can be excited by near-infrared light and emit visible light or ultraviolet light. Some studies have shown that under low-dose near-ultraviolet light (365 nm) irradiation, the secretion of extracellular exosomes will increase significantly, up to 13 times. However, the penetration of ultraviolet light is weak, while near-infrared light with a wavelength of 700-1000 nm has strong tissue penetration and higher safety, and will not cause obvious damage to tissues and cells.

[0005] Loading upconversion nanoparticles into mesenchymal stem cells, on the one hand, mesenchymal stem cells themselves have the ability to proliferate and differentiate into other cells, promoting immunomodulation and extracellular matrix remodeling; on the other hand, photo-controlled stimulation of mesenchymal stem cells releases more exosomes, and exosomes cooperate with mesenchymal stem cells to jointly treat tissue damage, significantly improving the effectiveness of treatment. Summary of the Invention

[0006] The purpose of the present invention is to provide a mesenchymal stem cell with a "photo-switch" effect (i.e., photo-responsive type), which can produce a large number of exosomes under the irradiation of a relatively safe light wavelength, thereby promoting the differentiation and maturation of osteoblasts and promoting bone injury repair.

[0007] In view of this, the solution of the present invention is as follows:

[0008] The first aspect of the present invention is to provide photo-responsive mesenchymal stem cells, which are obtained by incubating bone marrow mesenchymal stem cells in an upconversion nanoparticle solution.

[0009] Furthermore, the concentration of the upconversion nanoparticle solution is 1-100 μg / mL, preferably 20-500 μg / mL, and more preferably 50 μg / mL.

[0010] Furthermore, the upconversion nanoparticles are constructed with a rare earth material of NaYF4: 20% Yb, 0.5% Tm as the matrix.

[0011] The second aspect of the present invention is to provide a method for preparing exosomes. Near-infrared light is irradiated on a culture system containing the photo-responsive mesenchymal stem cells described in the first aspect, and the supernatant is collected and purified to obtain exosomes.

[0012] Furthermore, the wavelength of the near-infrared light is 980 nm.

[0013] Furthermore, the irradiation power of the near-infrared light is 1-50 W, the irradiation height is 0-50 cm, and the irradiation time is 1-45 min; preferably, the light irradiation power is 1 W, the irradiation height is 20 cm, and the irradiation time is 30 min.

[0014] Preferably, the collection time of the supernatant is 1-72 h after the near-infrared light irradiation, and more preferably 72 h.

[0015] The third aspect of the present invention is to provide the exosomes obtained by the preparation method described in the second aspect.

[0016] The fourth aspect of the present invention is to provide a bone injury repair agent, which includes the photo-responsive mesenchymal stem cells described in the first aspect, or the exosomes obtained by the preparation method described in the second aspect, or the exosomes described in the third aspect.

[0017] The fifth object of the present invention is to provide the application of the photo-responsive mesenchymal stem cells described in the first aspect above, or the exosomes described in the third aspect in the preparation of a bone injury repair agent.

[0018] Furthermore, the bone injury repair agent is used to promote the osteogenic differentiation or maturation of osteoblasts.

[0019] Furthermore, the bone injury repair includes, but is not limited to, the prevention and treatment of diseases such as fracture healing and regeneration, bone mass maintenance, and osteoporosis.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The photosensitive mesenchymal stem cells provided by the present invention have the ability to secrete a large amount of exosomes under near-infrared light illumination, with better safety, and can thus be used to prepare bone injury repair agents, providing a new option for drug compositions related to bone defect reconstruction.

[0022] The photosensitive mesenchymal stem cells of the present invention are simply prepared, have stable upconversion luminescence properties, and the combined treatment of exosomes and mesenchymal stem cells for tissue injury can significantly improve the effectiveness of treating bone tissue injury. Brief Description of the Drawings

[0023] Figure 1 It is a schematic diagram of the relevant characterization of the BMSC extraction and UCNPs preparation process of the present invention; wherein: Figure 1 A shows the successful induction of primary extracted BMSCs, co-expressing CD44 and CD90; Figure 1 B shows the emission spectrum of UCNPs; Figure 1 C shows the biocompatibility results of UCNPs on BMSCs detected by CCK8.

[0024] Figure 2 It is the verification result of the photosensitive performance of the prepared BMSC-UCNPs of the present invention; wherein: Figure 2 A shows the intracellular image of UCNPs in BMSCs under a biological electron microscope; Figure 2 B shows the emission spectrum of BMSC-UCNPs irradiated with a 980nm laser; Figure 2 C shows the quantitative influence results of different laser irradiation times on the extracted exosomes; Figure 2 D shows the quantitative influence results of different laser irradiation heights on the extracted exosomes; Figure 2 E shows the image of "light switch" promoting exosome release shown by a biological electron microscope, with the left being the control group and the right being the experimental group.

[0025] Figure 3 It is the result of BMSC-UCNPs releasing exosomes based on the photosensitive effect to promote osteogenic differentiation and maturation in vitro; wherein: Figure 3 A shows the EDU staining image of BMSC cells; Figure 3 B shows the data analysis result of EDU staining by Image J; Figure 3 C shows the ALP staining and alizarin red staining images of MC3T3-E1 cells; the upper figure is the ALP staining image, and the lower figure is the alizarin red staining image; Figure 3 D shows the data analysis result of ALP staining of MC3T3-E1 cells by Image J; Figure 3 E shows the data analysis result of alizarin red staining of MC3T3-E1 cells by Image J.

[0026] Figure 4PET-CT imaging results of the release of exosomes in vivo based on the photosensitive effect of BMSC-UCNPs of the present invention.

[0027] Figure 5 Results of promoting bone defect repair in vivo by BMSC-UCNPs of the present invention based on the photosensitive effect for 8 weeks. Detailed implementation manners

[0028] The technical solutions of the present invention will be clearly and completely described below in conjunction with the preferred embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] Example 1 Extraction and identification of bone marrow mesenchymal stem cells (BMSC)

[0030] BMSC cells were extracted from the femurs and tibias of 3- to 4-week-old SD rats.

[0031] 1) 3- to 4-week-old SD rats were sacrificed by cervical dislocation and immersed in 75% alcohol for 5 min.

[0032] 2) The rats were placed in a laminar flow hood, and the dissected and cleaned femurs and tibias were each added with a little PBS to submerge the tissues. The two ends of the epiphyses were cut open with scissors. After exposing the bone marrow cavity, a 5 mL syringe filled with PBS was used to flush the bone marrow cavity until the bone marrow cavity turned white.

[0033] 3) The flushed bone marrow was ground on a 45 μm filter membrane, repeatedly rinsed with PBS, and the filtered cell suspension was collected and centrifuged at 350 g for 5 min.

[0034] 4) After centrifugation, the supernatant was discarded, the cells were resuspended with complete medium, seeded in a 10 cm cell culture dish, and incubated at 37°C in 5% CO 2 overnight in a constant temperature incubator and the medium was changed the next day.

[0035] 5) After about 7-9 days, BMSC cells gradually took shape.

[0036] 6) The obtained cells were co-incubated with Anti-CD44 and Anti-CD90, and the positive cell staining results were observed under a confocal microscope. As Figure 1 shown in A, the extracted cells co-expressed CD44 and CD90, indicating successful induction of BMSC.

[0037] Example 2 Preparation and characterization of "light-switch" stem cells

[0038] 1. Preparation and characterization of upconversion nanoparticles

[0039] Construct upconversion nanoparticles (UCNPs) using a rare earth material with a matrix of NaYF4: 20% Yb, 0.5% Tm. Take the upconversion nanoparticles and prepare a 50 μg / mL solution with PBS. Detect the emission spectrum of the upconversion nanoparticles using a UCL fluorescence spectrometer. As Figure 1 shown in B, there is ultraviolet light emission at around 365 nm in the ultraviolet region.

[0040] 2. Biocompatibility of UCNPs with BMSCs

[0041] Seed BMSCs at a density of 5×10 3 cells / well in a 96-well plate. After culturing for 24 hours, add UCNPs solutions at concentrations of 0 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 500 μg / mL respectively, and continue culturing for 48 hours. Then, detect cell viability using CCK8. As Figure 1 shown in C, the maximum safe concentration of UCNPs is 50 μg / mL.

[0042] 3. Preparation of BMSC-UCNPs

[0043] 1) Prepare UCNPs at concentrations of 10 μg / mL, 20 μg / mL, 40 μg / mL, and 50 μg / mL respectively, and co-incubate them with BMSCs. Culture at 37 °C and 5% CO 2 for 24 hours. Add trypsin to digest the cells, centrifuge to remove the supernatant, wash twice with PBS, and finally resuspend the cells with PBS to obtain BMSC-UCNPs.

[0044] 2) Co-incubate 50 μg / mL of UCNPs and BMSCs for 24 hours. Digest the cells with trypsin, centrifuge to remove the cell supernatant, add 1 mL of 2.5% glutaraldehyde fixative at room temperature to the cell pellet, and fix at room temperature in the dark for 15 min. Observe the situation of UCNP inside BMSC cells under a biological electron microscope. As Figure 2 shown in A, UCNPs are effectively taken up by BMSCs and mainly exist in lysosomes, indicating the successful preparation of BMSC loaded with UNCP.

[0045] Example 3 BMSC-UCNPs Promote Exosome Release and Its Osteogenic Application Based on "Light Switch"

[0046] 1. "Light Switch" Effect of BMSC-UCNPs

[0047] BMSC-UCNPs with UCNP concentrations of 10 μg / mL, 20 μg / mL, 40 μg / mL, and 50 μg / mL were added to trypsin-digested cells. The supernatant was removed by centrifugation, and the cells were washed twice with PBS. Finally, the BMSC-UCNPs were resuspended in PBS. The suspension was injected into a quartz cell with a 1.5-mm slit, and the emission light intensity was measured under 980-nm excitation; as Figure 2 shown in B, 980-nm laser irradiation serves as the "optical switch" for UCNPs. As the concentration of UCNPs increases, the emission light intensity gradually increases.

[0048] 2. BMSC-UCNPs promote exosome release based on the "optical switch" effect

[0049] When BMSC cells grew to about 80%, complete medium containing 50 μg / mL of UCNPs was added. After 24 hours, the culture medium was aspirated, and the cells were washed twice with PBS. Then, 3 mL of complete medium was added, and a 980-nm laser was irradiated at a height of 20 cm from the cells for 0, 5, 10, 20, 30, and 45 min, respectively. The laser power was 1 W. After irradiation, complete medium was added to continue culturing the cells. After 72 hours, the cell supernatants of each group were collected, and exosomes were extracted and purified using a kit. The protein concentration of the exosomes was detected using a BCA kit for quantification. The results are shown in Figure 2 C. Laser irradiation at different times could promote exosome release, and the exosome release was the highest after 30-min irradiation. After determining that the irradiation time of 30 min was the optimal irradiation duration, the irradiation height was changed, and the irradiation heights were set to 0 cm, 10 cm, and 20 cm, respectively. The laser power was 1 W. After irradiation, complete medium was added to continue culturing the cells. After 72 hours, the same quantification was performed on the extracted exosomes. The results are shown in Figure 2 D. The exosome release was the highest in the group irradiated at a height of 20 cm. Therefore, the laser irradiation height was finally determined to be 20 cm, and the irradiation time was 30 min.

[0050] Based on the conditions explored above, the situation of exosome release promoted by the optical switch was further observed using a biological electron microscope, as shown in Figure 2 E. Compared with the control group, more exosomes could be released extracellularly from mesenchymal stem cells irradiated with a 980-nm laser "optical switch".

[0051] 3. Osteogenic application of exosomes released by BMSC-UCNPs based on the "optical switch" effect

[0052] BMSC cells were seeded at 5×10 per well 3Inoculated into 96-well plates, after 24 h, complete media containing 0 μg / mL, 10 μg / mL, 20 μg / mL, and 30 μg / mL of BMSC exosomes (BMSC-Exo) were added respectively. After 48 h, 100 μL of 50 μM EDU medium was added to each well and incubated for 2 h. The cell nuclei were stained with hoechst33342. As Figure 3 shown in A and 3B, compared with the control group, exosomes at different concentrations could promote the proliferation of BMSC cells. Further, osteoblasts MC3T3-E1 were evenly inoculated into 24-well plates, with 2×10 4 cells per well, and co-incubated with 0 μg / mL, 10 μg / mL, 20 μg / mL, and 30 μg / mL of BMSC exosomes (BMSC-Exo) respectively. After 48 h, the medium containing exosomes was aspirated, and the cells were washed twice with PBS; MC3T3-E1 osteogenic induction medium was added and cultured for 14 days and 21 days respectively, followed by ALP and alizarin red staining; as Figure 3 shown in C, 3D, and 3E, BMSC-Exo at different concentrations could stimulate the osteogenic differentiation and maturation of osteoblasts.

[0053] 4. In vivo "light-switch" effect and osteogenic application of BMSC-UCNPs

[0054] BMSC-UCNPs were labeled with azide-modified mannose and administered to rats with cranial bone defects. Based on the tail vein injection of radionuclide modified with DBCO at the defect site, PET-CT imaging was performed. It was found that BMSC-UCNPs also had a "light-switch effect" in vivo, and the light signal at the cranial bone defect site was significantly enhanced, indicating that more exosomes of MSCs were released in vivo ( Figure 4 ). Further, BMSC-UCNPs were administered to rats with cranial bone defects in vivo. It was found that combined with 980 nm laser irradiation, bone defect repair was significantly promoted after 8 weeks ( Figure 5 ).

[0055] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Light-sensitive mesenchymal stem cells, characterized in that: Obtained by incubating bone marrow mesenchymal stem cells in a solution of upconversion nanoparticles.

2. The light-sensitive mesenchymal stem cells according to claim 1, characterized in that The concentration of the up-conversion nanoparticle solution is 1-100 μg / mL; And / or, the up-conversion nanoparticles are constructed using a rare earth material of NaYF4: 20% Yb, 0.5% Tm as a matrix.

3. A method for preparing exosomes, characterized in that: Irradiating the culture system containing the light-sensitive mesenchymal stem cells according to claim 1 with near-infrared light, collecting the supernatant and purifying it to obtain exosomes.

4. The preparation method according to claim 3, characterized in that: The wavelength of the near infrared light is 980 nm.

5. The preparation method according to claim 3, characterized in that: The near-infrared light irradiation power is 1-50W, the irradiation height is 0-50cm, and the irradiation time is 1-45min.

6. The preparation method according to claim 5, characterized in that: The supernatant is collected for 1-72 hours after near-infrared light irradiation.

7. Exosomes obtained by the preparation method according to any one of claims 3 to 6.

8. A bone damage repair agent, characterized in that: It comprises the light-sensitive mesenchymal stem cells according to claim 1 or 2, or the exosomes according to claim 7.

9. Use of the light-sensitive mesenchymal stem cells according to claim 1 or 2, or the exosomes according to claim 7 in the preparation of a bone damage repair agent.

10. The use according to claim 9, characterized in that: The bone damage repair agent is used for promoting osteoblast osteogenic differentiation or maturation.

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