A nested microsphere hydrogel carrying dual exosomes and its preparation method and application

Abstract

The present invention is applicable to the field of tissue engineering technology and provides a preparation method of a nested microsphere hydrogel carrying dual exosomes, comprising: preparing o-exos and exos; preparing a white porous foam GelMA hydrogel precursor; weighing Fibrinogen and dissolving it in PBS containing o-exos to obtain a Fibrinogen hydrogel precursor solution carrying exosomes; and performing photocrosslinking polymerization to obtain Fibrinogen. o‑exos Microsphere hydrogel; GelMA hydrogel precursor was weighed and dissolved in PBS containing exos, and then mixed with Fibrinogen o‑exos The microsphere hydrogel is fully mixed and then photocrosslinked to obtain GelMA. exos ‑Fibrinogen o‑exos Nested microsphere hydrogel. The present invention also provides a nested microsphere hydrogel loaded with dual exosomes. The present invention also provides the use of the nested microsphere hydrogel loaded with dual exosomes in the preparation of an osteogenic material for the maxillary sinus region. The present invention uses a water-oil emulsification method to prepare a dual sustained-release exosome nested microsphere hydrogel, providing a theoretical basis for in situ bone regeneration in the treatment of maxillary sinus defects.

Description

Technical Field

[0001] The present invention belongs to the field of tissue engineering technology, and in particular relates to a nested microsphere hydrogel carrying dual exosomes, a preparation method, and an application thereof. Background Art

[0002] After maxillary posterior tooth loss, alveolar bone resorption and maxillary sinus pneumatization result in insufficient residual bone height, increasing the risk and difficulty of implant surgery. In recent years, sinus floor augmentation has gradually become a key clinical technique for addressing vertical bone height deficiency in the maxillary posterior region and has been accepted and applied worldwide. Literature reports indicate that sinus floor augmentation without bone grafting can result in significant new bone formation within the sinus floor space. Studies have found that centripetally growing new woven bone and medullary cavities can be observed within the sinus floor mucosa after augmentation. This suggests that the maxillary sinus mucosa, also known as Schneider's membrane, not only maintains the stability of the osteoblastic space but also contains mesenchymal stem cells with osteogenic potential. Subsequent studies have successfully isolated and identified mesenchymal stem cells with osteogenic potential within the Schneider's membrane. However, the specific mode and mechanism of maxillary sinus osteogenesis remain controversial. What is certain is that developing efficient in situ autologous bone regeneration strategies for the maxillary sinus based on mesenchymal stem cells to restore residual bone height in the maxillary sinus is crucial.

[0003] Exosomes are natural nanovesicles with a diameter of approximately 40-150 nm that originate from endosomes. They have many advantages, such as a wide range of sources, the ability to artificially modify and change the quantity and type of contents, and the ability to avoid distortion caused by excessive proliferation of stem cells. They play an important role in intercellular material exchange and signal communication, and have the potential to replace mesenchymal stem cells in applications.

[0004] Exosomes derived from various stem cell sources have been well-established to play roles in bone repair, including modulating immune responses, inhibiting bone resorption, recruiting and inducing mesenchymal stem cells (MSCs), creating an osteoinductive environment to promote osteogenic differentiation, and inducing angiogenesis. Inspired by this, exosomes derived from Schneider's membrane-derived MSCs were used. While avoiding immunogenic responses caused by direct cell-cell contact, they were able to deliver biomolecules to target cells, promoting signaling between MSCs in the maxillary sinus. However, Schneider's membrane-derived MSCs exhibited limited migration and osteogenic capacity, and their numbers were relatively low. Leveraging the ability of exosomes to alter their contents in response to natural environments, two engineered exosomes were extracted and modified by altering the tissue microenvironment in which cells survive. These exosomes, selectively expressed according to their gene expression profiles, enable a phased approach to osteogenic differentiation, potentially promoting a more organized progression of stem cell migration and osteogenesis.

[0005] When applied directly in vivo, exosomes are easily captured by the reticuloendothelial system and phagocytosed by monocytes, resulting in limited targeting and low local effective concentrations. Currently, the use of bioscaffolds to prolong the storage and release of exosomes is widely accepted and applied. Hydrogels, with their excellent biocompatibility and similarity to the tissue microenvironment, are being used as scaffold materials to maintain the osteoblastic space. Currently, most approaches involve directly loading natural exosomes into osteoblastic scaffolds to promote bone regeneration. However, the function of individual exosomes is limited, and the loading method and scaffold material selection remain under investigation. Due to limitations in the maxillary sinus anatomy and difficulties in cell culture, there are currently few reports on the application of exosome-loaded hydrogels in maxillary sinus bone defects. To maximize the diverse benefits of stem cells during the osteogenesis phase, improved scaffolds are urgently needed. The fluidity and injectability of injectable hydrogels and microsphere hydrogels meet the requirements for bone grafting in the maxillary sinus. Theoretically, by improving the diameter of the microspheres, constructing a nested microsphere structure, and utilizing the encapsulation properties of the microsphere hydrogel, effective loading of exosomes with different functions can be achieved. In addition, by artificially controlling the gradual release of exosomes during the degradation of the hydrogel, spatiotemporal regulation of stem cell function at different stages of bone regeneration can be achieved.

[0006] The application of nested microsphere hydrogels loaded with exosomes has sufficient theoretical basis for achieving in situ bone regeneration in maxillary sinus bone defects and is expected to achieve innovative breakthroughs. Summary of the Invention

[0007] The purpose of the embodiments of the present invention is to provide a method for preparing a nested microsphere hydrogel carrying dual exosomes, aiming to solve the problems raised in the above background technology.

[0008] The present invention is implemented as follows: a method for preparing a nested microsphere hydrogel carrying dual exosomes comprises the following steps:

[0009] Step 1: preparing exosomes o-exos derived from Schneider membrane mesenchymal stem cells under osteogenic induction conditions and exosomes exos derived from Schneider membrane mesenchymal stem cells under non-osteogenic induction conditions;

[0010] Step 2: Prepare a white porous foamy GelMA hydrogel precursor using type A pigskin gelatin;

[0011] Step 3: Weigh Fibrinogen and dissolve it in sterile PBS containing exosome o-exos concentrate to obtain a Fibrinogen hydrogel precursor solution loaded with exosomes. Add a photoinitiator and perform photocrosslinking polymerization under ultraviolet light to obtain Fibrinogen. o-exos microsphere hydrogel;

[0012] Step 4: Weigh the GelMA hydrogel precursor and dissolve it in sterile PBS containing exosome concentrate, and then add Fibrinogen o-exos The microsphere hydrogel is fully mixed to obtain GelMA exos -Fibrinogen o-exos The hydrogel precursor solution is added with a photoinitiator and photocrosslinked and polymerized under ultraviolet light to obtain GelMA. exos -Fibrinogen o-exos Nested microsphere hydrogel.

[0013] Another object of an embodiment of the present invention is to provide a nested microsphere hydrogel carrying dual exosomes, which is prepared by the above preparation method.

[0014] Another object of an embodiment of the present invention is to provide a use of the above-mentioned nested microsphere hydrogel carrying dual exosomes in the preparation of bone osteogenic materials in the maxillary sinus area.

[0015] The present invention provides a method for preparing a nested microsphere hydrogel carrying dual exosomes. The method uses two naturally low-immunogenic protein materials, gelatin methacryloyl (GelMA) and fibrinogen (Fibrinogen), and applies them to bone osteogenic materials in the maxillary sinus region. As scaffold materials for bone tissue engineering, they can reduce the immune response after scaffold implantation. At the same time, GelMA and Fibrinogen are also photoresponsive hydrogel materials. Photoresponsive hydrogels have few byproducts and good biocompatibility, and have great application prospects in bone tissue engineering applications. Photocrosslinked microspheres made of GelMA as a raw material have been commercialized, but they degrade slowly and have little effect on cell migration, proliferation, and differentiation. Fibrinogen has good cell adhesion and recruitment functions, which can to some extent make up for the shortcomings of GelMA microsphere hydrogel applications. Under the action of a photocrosslinker, the Fibrinogen and GelMA precursor solutions are chemically crosslinked respectively. The microsphere diameter is changed by adjusting the rotation speed to synthesize GelMA nested microspheres encapsulated with Fibrinogen, providing a structural basis for the subsequent step-by-step release of exosomes with different functions.

[0016] Schneider membrane stem cells cultured under two different conditions were ultracentrifuged, and the two exosomes extracted were loaded into Fibrinogen and GelMA microspheres, respectively. Leveraging the low immunogenicity of exosomes, the researchers improved the biosafety of the microsphere hydrogels after implantation into the maxillary sinus bone defect. The interaction between the exosomes and the microsphere hydrogels controlled and prolonged the exosomes' retention time within the microspheres, enabling a stable and continuous, step-by-step release of the exosomes during the layered degradation of the nested microspheres. These exosomes induced the proliferation, migration, and osteogenic differentiation of Schneider membrane-derived mesenchymal stem cells, achieving biological integration of cell migration, proliferation, and osteogenic differentiation around the maxillary sinus bone wall, and accelerating in situ bone regeneration in the maxillary sinus region.

[0017] The dual sustained-release exosome-nested microsphere hydrogel prepared by the water-oil emulsification method in the embodiment of the present invention provides a theoretical basis for the in situ bone regeneration in the treatment of maxillary sinus defect areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Osteo-SMMSCs provided by the embodiment of the present invention exos TEM images of

[0019] Figure 2 SMMSCs provided by the embodiment of the present invention exos TEM images of

[0020] Figure 3 Osteo-SMMSCs provided by the embodiment of the present invention exos Particle size analysis results;

[0021] Figure 4 SMMSCs provided by the embodiment of the present invention exos Particle size analysis results;

[0022] Figure 5 Osteo-SMMSCs provided by the embodiment of the present invention exos and SMMSCs exos Western blot analysis results of surface markers;

[0023] Figure 6 OM image of the nested microsphere hydrogel provided by an embodiment of the present invention;

[0024] Figure 7 This is an SEM image of the nested microsphere hydrogel provided by an embodiment of the present invention;

[0025] Figures 8A-8E Transwell cell migration assay results provided by the present invention ( Figures 8A-8E Blank group, GelMA-Fibrinogen, GelMAexos -Fibrinogen, GelMA-Fibrinogen o-exos 、GelMA exos -Fibrinogen o-exos );

[0026] Figures 9A-9E The alkaline phosphatase staining results provided by the embodiment of the present invention show ( Figures 9A-9E Blank group, GelMA-Fibrinogen, GelMA exos -Fibrinogen, GelMA-Fibrinogen o-exos 、GelMA exos -Fibrinogen o-exos );

[0027] Figures 10A-10E The hematoxylin and eosin (HE) staining results provided in the embodiment of the present invention (New Bone, referred to as NB, original bone Basal Bone) ( Figures 10A-10E Blank group, GelMA-Fibrinogen, GelMA exos -Fibrinogen, GelMA-Fibrinogen o-exos 、GelMA exos -Fibrinogen o-exos );

[0028] Figures 11A-11E The Micro-CT results provided by the embodiment of the present invention ( Figures 11A-11E Blank group, GelMA-Fibrinogen, GelMA exos -Fibrinogen, GelMA-Fibrinogen o-exos 、GelMA exos -Fibrinogen o-exos );

[0029] Figure 12 Flow chart of exosome extraction provided in an embodiment of the present invention;

[0030] Figure 13 Flowchart for synthesizing a porous foam-like GelMA hydrogel precursor provided by an embodiment of the present invention;

[0031] Figure 14 Synthetic GelMA provided by the embodiment of the present invention exos -Fibrinogen o-exos Flowchart of nested microsphere hydrogels. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0033] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0034] Example 1

[0035] A method for preparing a nested microsphere hydrogel carrying dual exosomes comprises the following steps:

[0036] Extraction of exosomes, such as Figure 12 As shown, specifically including:

[0037] (1) The fourth generation human Schneider membrane mesenchymal stem cells were cultured at 1×10 6 The cells were seeded at a density of 1 / mL in α-MEM medium containing 10% fetal bovine serum by volume and cultured at 37°C and 5% CO2 until the density reached 90%. The culture medium was then replaced with exosome-free serum medium or exosome-free serum medium supplemented with osteogenic induction components. After 48 hours, the cell supernatant was collected in a 50 mL centrifuge tube, and the remaining cells were passaged or discarded according to the cell status.

[0038] (2) Centrifuge at 300 g for 10 min at 4°C, discard the remaining cells in the pellet, and transfer the supernatant in the centrifuge tube to a new 50 mL centrifuge tube;

[0039] (3) Centrifuge at 2000 g for 10 min at 4°C, discard the necrotic cells in the pellet, and transfer the supernatant in the centrifuge tube to a new 50 mL centrifuge tube;

[0040] (4) Centrifuge at 6000 g for 30 min at 4°C to remove cell debris and organelles from the pellet. Transfer the supernatant from the centrifuge tube to a new 50 mL centrifuge tube.

[0041] (5) Centrifuge at 16500 g for 30 min at 4°C to further precipitate the cell debris in step (4), and transfer the supernatant in the centrifuge tube to a new 50 mL centrifuge tube;

[0042] (6) Transfer the cell supernatant from step (5) to an ultrafiltration centrifuge tube, centrifuge at 4000 g for 40 min, gently mix the supernatant concentrate in the cell concentration tank with a 200 μl pipette, pass the concentrate through a 0.22 μm syringe filter, and transfer it to a new ultrafiltration centrifuge tube using a 1 mL syringe;

[0043] (7) Centrifuge at 100,000 g for 70 min at 4°C, discard the supernatant with a pipette, add 200 μl of sterile PBS, and slowly resuspend the precipitate to dissolve it;

[0044] (8) The resuspended solution was transferred to a new ultrafiltration centrifuge tube, and sterile PBS was slowly added to the neck of the tube. Centrifuge at 100,000 g for 70 min at 4 °C, discard the supernatant, and add 100 μl of sterile PBS to resuspend the precipitate. Finally, the osteogenic induction condition (Osteo-SMMSCs) was obtained. exos , hereinafter abbreviated as o-exos) and under non-osteoinductive conditions (SMMSCs exos Exosomes derived from Schneider's membrane mesenchymal stem cells (hereinafter referred to as exos) were placed at 4°C for later use and stored at -80°C for long-term storage;

[0045] Synthesize porous foam-like GelMA hydrogel precursors, such as Figure 13 As shown, specifically including:

[0046] (1) 7.5 g of type A pigskin gelatin and 75 mL of sterile PBS were added to a conical flask containing 75 mL of sterile PBS in small amounts and several times. The mixture was placed in a 50°C water bath and stirred continuously for 1-2 h until the liquid in the conical flask became light yellow and clear. At this time, the type A pigskin collagen was completely dissolved, thereby obtaining a GelMA hydrogel precursor solution with a concentration of 10% w / v;

[0047] (2) 750 μl of methacrylic anhydride was slowly added dropwise to the continuously stirred GelMA hydrogel precursor solution at a rate of 1 mL / min, and the reaction was continued for 3 h.

[0048] (3) After 3 h, the pH of the GelMA hydrogel precursor solution was adjusted to neutral or near neutral. The GelMA hydrogel precursor solution after the reaction was stopped was transferred to a dialysis bag with a retention rate of 8000-14000. The GelMA hydrogel precursor solution was dialyzed in 50°C distilled water for 2 weeks. The distilled water was replaced every 4 h in the first week and every 8 h in the second week.

[0049] (4) Centrifuge the dialyzed GelMA hydrogel precursor solution at 6000-7000 rpm for 5 min at room temperature using a filter membrane;

[0050] (5) After centrifugation, the supernatant was aspirated into a 10 cm culture dish, cooled to a jelly-like state, and placed in a -80°C refrigerator overnight;

[0051] (6) The pre-frozen GelMA hydrogel precursor solution was taken out from the -80°C refrigerator and placed in a freeze dryer for 48 h to obtain a white porous foam-like GelMA hydrogel precursor;

[0052] Synthesis of GelMA exos -Fibrinogen o-exos Nested microsphere hydrogels, e.g. Figure 14 As shown, specifically including:

[0053] (1) Weigh 50 mg of Fibrinogen and dissolve it in 1 mL of sterile PBS containing exosome o-exos concentrate to obtain a 5% Fibrinogen hydrogel precursor solution loaded with exosomes. Add 2 mg of photoinitiator and perform photocrosslinking polymerization under 405 nm ultraviolet light for 5 min to obtain Fibrinogen. o-exos microsphere hydrogel;

[0054] (2) Weigh 100 mg of GelMA hydrogel precursor and dissolve it in 1 mL of sterile PBS containing exosome concentrate, and then add 10 mg of Fibrinogen o-exos The microsphere hydrogel is fully mixed to obtain GelMA exos -Fibrinogen o -exos The hydrogel precursor solution was added with 2 mg of photoinitiator and photocrosslinked and polymerized under 405 nm UV light for 5 min to obtain GelMA. exos -Fibrinogen o-exos Nested microsphere hydrogel.

[0055] Identification, analysis and results:

[0056] 1. Identification of exosomes derived from human Schneider membrane mesenchymal stem cells:

[0057] Osteo-SMMSCs were characterized by transmission electron microscopy (TEM). exo and SMMSCs exos Observe and get the results as Figure 1-4 As shown, it can be seen that Osteo-SMMSCs exos and SMMSCs exos Round cup-shaped structure ( Figure 1 、 Figure 2 ), with complete membrane coating and scattered distribution, Osteo-SMMSCs exos and SMMSCs exos The particle size range of the two exosomes is mainly between 30-150nm ( Figure 3 、 4 );

[0058] Osteo-SMMSCs exos and SMMSCs exosWestern blot (WB) analysis of surface markers showed that Figure 5 As shown, the specific proteins CD9(+), CD81(+), and TSG101(+) were detected, and the precipitates extracted by ultracentrifugation were indeed exosomes.

[0059] 2. Microstructure characterization of nested microspheres:

[0060] The nested microspheres prepared in Example 1 were observed under a common optical microscope. The obtained images were processed by Image J software and the particle sizes of the microspheres were statistically analyzed. The results were as follows: Figure 6 As shown;

[0061] The appearance and particle size distribution of the microspheres were characterized using SEM. A small amount of microspheres were evenly distributed on the conductive adhesive and placed in a gold spraying instrument for 15 minutes to enhance the conductivity of the microspheres. The microspheres were placed in a scanning electron microscope and the fine structure of the microsphere surface was observed under vacuum conditions. The size and distribution of the microspheres were statistically analyzed using software. The results are as follows: Figure 7 As shown;

[0062] As can be seen from the figure, the microspheres are regular spherical, with good morphology and no obvious adhesion. The particle size of the GelMA microsphere hydrogel is about 60-150 μm, and the particle size of the Fibrinogen microsphere hydrogel is about 5-20 μm.

[0063] 3. Migration of Schneider Membrane-Derived Mesenchymal Stem Cells Induced by Nested Microsphere Hydrogels Loaded with Exosomes:

[0064] The Transwell migration assay was used to measure cell migration ability. The Transwell chamber divides the well plate into an upper chamber and a lower chamber. The upper chamber contains cells, and the lower chamber contains nested microsphere hydrogels. The upper and lower chambers are separated by a porous membrane that allows cells to pass through. The migration ability between different materials is measured by the number of cells passing through the porous membrane.

[0065] To detect GelMA exos -Fibrinogen o-exos Whether the nested microsphere hydrogel has a driving effect on Schneider membrane-derived mesenchymal stem cells, this experiment used a 6-well plate Transwell system (8um pore size) to conduct a cell migration experiment. The experiment was divided into 5 groups, with the no microsphere group (cells were seeded only in the upper chamber) as the blank group, the simple GelMA-Fibrinogen nested microspheres as the control group, and the experimental group with GelMA exos -Fibrinogen, GelMA-Fibrinogen o-exos , GelMA exos -Fibrinogen o-exos , the specific experimental steps are as follows:

[0066] 1) Take Schneider membrane mesenchymal stem cells P4 in the logarithmic growth phase, remove the old culture medium, and wash with PBS three times;

[0067] 2) Add 0.25% trypsin for digestion. After the cells shrink and become round, add α-MEM medium to terminate the digestion reaction.

[0068] 3) Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes at room temperature;

[0069] 4) Resuspend the cells in culture medium and count the cells to adjust the cell concentration to 5x10 5 100 μl of cell suspension was added to the upper chamber of the Transwell, and microsphere hydrogels of different groups were placed in the lower chamber;

[0070] 5) Incubate the plate in a 37°C 5% CO2 incubator for 24-48 hours;

[0071] 6) Aspirate the culture medium and fix with 4% paraformaldehyde for 15 minutes;

[0072] 7) Wash with PBS three times and remove the unmigrated cells in the upper chamber with a cotton swab;

[0073] 8) Crystal violet staining for 20-30 minutes, then gently rinse with PBS three times;

[0074] 9) Observe the results under an inverted optical microscope;

[0075] Transwell cell migration assay results Figures 8A-8E As shown, compared with the control group, GelMA exos -Fibrinogen and GelMA exos -Fibrinogen o-exos The two groups could significantly promote the migration of Schneider membrane-derived stem cells in the maxillary sinus, and there was no significant difference between the two groups. o-exos The ability of the group to induce cell migration was weaker, but better than that of the control group and the blank group.

[0076] 4. Exosome-loaded nested microsphere hydrogels induce osteogenic differentiation of Schneider membrane-derived mesenchymal stem cells:

[0077] To detect GelMA exos -Fibrinogen o-exosWhether microsphere hydrogel has osteoinductive effect on Schneider membrane-derived mesenchymal stem cells, this experiment used a 6-well plate Transwell system (8um pore size) for microsphere-cell non-contact co-culture. The experiment was divided into 5 groups, with the no microsphere group (cells seeded only in the upper chamber) as the blank group, the simple GelMA-Fibrinogen group as the control group, and the experimental group with GelMA exos -Fibrinogen, GelMA-Fibrinogen o-exos , GelMA exos -Fibrinogen o-exos , the specific experimental steps are as follows:

[0078] 1) Take Schneider membrane mesenchymal stem cells P4 in the logarithmic growth phase, remove the old culture medium, and wash with PBS three times;

[0079] 2) Add 0.25% trypsin for digestion. After the cells shrink and become round, add α-MEM medium to terminate the digestion reaction.

[0080] 3) Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes at room temperature;

[0081] 4) Resuspend the cells in culture medium and count the cells to adjust the cell concentration to 5x10 5 Add 100 μl of cell suspension to the lower chamber of a 6-well Transwell plate, add 2 mL of α-MEM medium to each well, shake gently to evenly spread the cells, and place in a 37°C 5% CO2 incubator overnight;

[0082] 5) After 24 hours, observe cell adhesion and replace the culture medium with osteogenic induction medium. Place the microsphere hydrogels from different experimental groups in the upper chamber. During the culture period, replace the osteogenic induction medium every 2-3 days.

[0083] 6) After 7 days of co-culture, discard the culture medium and rinse three times with 1-2 mL of PBS;

[0084] 7) Add 2 mL of 4% paraformaldehyde to each well and fix for 15 minutes. Rinse gently with PBS three times.

[0085] 8) As instructed, add 1 mL of BCIP / NBT alkaline phosphatase colorimetric working solution to each well, covering the cells on the bottom wall of the plate. Incubate at room temperature in the dark for 30 minutes. Aspirate the staining solution, wash with PBS, and observe and capture images under an inverted optical microscope.

[0086] After 7 days of culture, the alkaline phosphatase staining results were as follows: Figures 9A-9E As shown, compared with the blank group, GelMA exos -Fibrinogen o-exosGroup, GelMA-Fibrinogen o-exos Group, GelMA exos The colors of the -Fibrinogen and GelMA-Fibrinogen groups weakened in turn, and the results showed that the nested microsphere hydrogel loaded with exosomes could induce the osteogenic differentiation of Schneider membrane-derived mesenchymal stem cells.

[0087] 5. Effects of exosome-loaded nested microsphere hydrogel on bone tissue regeneration and repair in a rabbit maxillary sinus defect model:

[0088] Maxillary sinus bone defect models were established in 10 New Zealand white rabbits at 4 weeks old. The blank group, GelMA-Fibrinogen, GelMA exos -Fibrinogen, GelMA-Fibrinogen o-exos 、GelMA exos -Fibrinogen o-exos Five groups of microsphere hydrogels, each with 4 replicate wells, the specific operation steps are as follows:

[0089] 1) Preoperative preparation: sterilization of materials, preparation and sterilization of instruments, and fasting of animals from food and water for 12 hours before surgery;

[0090] 2) Preparation of maxillary sinus floor lift model: New Zealand white rabbits were weighed and anesthetized with an injection of Antai (0.2 mL / kg) into the gluteus maximus. The nasal dorsum was prepared and disinfected with iodine. After deiodination with 75% alcohol, a drape was laid and 0.5-1 mL of pyralid was injected into the nasal dorsum for local infiltration anesthesia. A vertical incision of approximately 2-3 cm in length was made along the midline of the nose and face. The skin was incised until the periosteum was completely peeled off with a dissector to fully expose the nasal bones and the bilateral nasal bone junctions. Under pre-cooled sterile saline irrigation, a trephine was used to drill 2 cm in front of the nasofrontal suture and 0.5 cm outside the midline, on each side. A 5mm diameter bone window was prepared. Sterile forceps were used to carefully remove the bone fragment, and the intact rabbit Schneider's membrane, which rose and fell with breathing, could be observed. A dissector was used to gently push the maxillary sinus mucosa upward and backward along the bone wall in accordance with the respiratory rhythm. The preoperatively disinfected material was filled into this space. The periosteum and mucosa were sutured and the incision was tightly sutured. The animals were kept warm after surgery and their condition was monitored daily. 600,000 units of penicillin were injected daily for 3 days after surgery. The rabbits were euthanized at 4, 8, and 12 weeks after surgery, and the rabbit maxillary sinus models were collected for imaging and histological analysis.

[0091] 3) Sectioning: Pre-cool the embedded tissue. Adjust the position and angle of the tissue with the specimen clamp and slice it to a thickness of 3-5 μm. Expand the paraffin sections in a 45°C water bath. Tilt the slide into the water surface and gently pick up the expanded sections. Remove excess water, mark the groups with a pencil, and bake the sections at 60°C for 2 hours until the paraffin disappears.

[0092] 4) HE staining: The sections were sequentially immersed in xylene I for 15 minutes and xylene II for 15 minutes for dewaxing. After dewaxing, they were immersed in anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 95% ethanol for 5 minutes, 90% ethanol for 5 minutes, and 80% ethanol for 5 minutes. Finally, they were rinsed with distilled water for 10 minutes. After hematoxylin staining for 5 minutes, the sections were removed and rinsed with plenty of tap water. After hydrochloric acid alcohol was removed for 10 seconds, the sections were rinsed with plenty of tap water, and the nuclear staining effect was observed under a microscope. After eosin staining for 2 minutes, the sections were removed and rinsed with plenty of tap water. The cytoplasmic staining effect was observed under a microscope. Finally, the sections were dehydrated and transparently mounted. The sections were sequentially immersed in 75% ethanol for 5 minutes, 80% ethanol for 5 minutes, 95% ethanol I for 5 minutes, 95% ethanol II for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes. After thorough air drying, the sections were mounted with neutral gum and air-dried.

[0093] 5) Micro-CT examination of bone tissue: The collected bone tissue specimens were fixed in 4% paraformaldehyde for 48 hours and rinsed with running water for 2 hours. The bone tissue was scanned and three-dimensionally reconstructed using Micro-CT. Bone volume (BV) was observed and analyzed, and statistical comparisons were performed.

[0094] The results of hematoxylin and eosin (HE) staining were as follows Figures 10A-10E As shown, the Micro-CT results are as follows Figures 11A-11E As described above, the bone defect healing was evaluated 4 weeks after maxillary sinus augmentation in rabbits. The results of HE staining and Micro-CT showed that the new bone formation in the blank group and the control group was almost negligible, while the GelMA exos -Fibrinogen o-exos The group had a large amount of matrix formation in the defect area, and the bone defect healed quickly with more new bone formation.

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a nested microsphere hydrogel carrying dual exosomes, characterized in that: The following steps are involved: Step 1: preparing exosomes o-exos derived from Schneider membrane mesenchymal stem cells under osteogenic induction conditions and exosomes exos derived from Schneider membrane mesenchymal stem cells under non-osteogenic induction conditions; Step 2: Prepare a white porous foamy GelMA hydrogel precursor using type A pigskin gelatin; Step 3: Weigh Fibrinogen and dissolve it in sterile PBS containing exosome o-exos concentrate to obtain a Fibrinogen hydrogel precursor solution loaded with exosomes. Add a photoinitiator and perform photocrosslinking polymerization under ultraviolet light to obtain Fibrinogen. o-exos microsphere hydrogel; Step 4: Weigh the GelMA hydrogel precursor and dissolve it in sterile PBS containing exosome concentrate, and then add Fibrinogen o-exos The microsphere hydrogel is fully mixed to obtain GelMA exos -Fibrinogen o-exos The hydrogel precursor solution is added with a photoinitiator and photocrosslinked and polymerized under ultraviolet light to obtain GelMA. exos -Fibrinogen o-exos Nested microsphere hydrogel.

2. The method for preparing the nested microsphere hydrogel carrying dual exosomes according to claim 1, characterized in that: The specific process of preparing exosomes (o-exos) derived from Schneider membrane mesenchymal stem cells under osteogenic induction conditions in step 1 includes: (1) The fourth generation human Schneider membrane mesenchymal stem cells were cultured at 1×10 6 The cells were seeded at a density of 1 / mL in α-MEM medium containing 10% fetal bovine serum by volume and cultured at 37°C and 5% CO2 until the density reached 90%. The culture medium was then replaced with an exosome-free serum medium supplemented with osteogenic induction components. After 48 hours, the cell supernatant was collected in a 50 mL centrifuge tube, and the remaining cells were passaged or discarded according to the cell status. (2) Centrifuge at 300 g for 10 min at 4°C, discard the remaining cells in the pellet, and transfer the supernatant in the centrifuge tube to a new 50 mL centrifuge tube; (3) Centrifuge at 2000 g for 10 min at 4°C, discard the necrotic cells in the pellet, and transfer the supernatant in the centrifuge tube to a new 50 mL centrifuge tube; (4) Centrifuge at 6000 g for 30 min at 4°C to remove cell debris and organelles from the pellet. Transfer the supernatant from the centrifuge tube to a new 50 mL centrifuge tube. (5) Centrifuge at 16500 g for 30 min at 4°C to further precipitate the cell debris in step (4), and transfer the supernatant in the centrifuge tube to a new 50 mL centrifuge tube; (6) Transfer the cell supernatant from step (5) to an ultrafiltration centrifuge tube, centrifuge at 4000 g for 40 min, gently mix the supernatant concentrate in the cell concentration tank with a 200 μl pipette, pass the concentrate through a 0.22 μm syringe filter, and transfer it to a new ultrafiltration centrifuge tube using a 1 mL syringe; (7) Centrifuge at 100,000 g for 70 min at 4°C, discard the supernatant with a pipette, add 200 μl of sterile PBS, and slowly resuspend the precipitate to dissolve it; (8) The resuspended solution was transferred to a new ultrafiltration centrifuge tube, and sterile PBS was slowly added to the neck of the tube. Centrifuged at 100,000 g for 70 min at 4 °C, the supernatant was discarded, and 100 μl of sterile PBS was added to resuspend the precipitate. Finally, exosomes derived from Schneider membrane mesenchymal stem cells under osteogenic induction conditions were obtained. exos , abbreviated as o-exos, placed at 4℃ for standby use and stored at -80℃ for long-term storage.

3. The method for preparing the nested microsphere hydrogel carrying dual exosomes according to claim 1, characterized in that: The specific process of preparing exosomes derived from Schneider membrane mesenchymal stem cells under non-osteogenic induction conditions in step 1 includes: (1) The fourth generation human Schneider membrane mesenchymal stem cells were cultured at 1×10 6 The cells were seeded at a density of 1 / mL in α-MEM medium containing 10% fetal bovine serum by volume, cultured at 37°C and 5% CO2 to a density of 90%, and then replaced with exosome-free serum medium. After 48 hours, the cell supernatant was collected in a 50 mL centrifuge tube, and the remaining cells were passaged or discarded according to the cell status; (2) Centrifuge at 300 g for 10 min at 4°C, discard the remaining cells in the pellet, and transfer the supernatant in the centrifuge tube to a new 50 mL centrifuge tube; (3) Centrifuge at 2000 g for 10 min at 4°C, discard the necrotic cells in the pellet, and transfer the supernatant in the centrifuge tube to a new 50 mL centrifuge tube; (4) Centrifuge at 6000 g for 30 min at 4°C to remove cell debris and organelles from the pellet. Transfer the supernatant from the centrifuge tube to a new 50 mL centrifuge tube. (5) Centrifuge at 16500 g for 30 min at 4°C to further precipitate the cell debris in step (4), and transfer the supernatant in the centrifuge tube to a new 50 mL centrifuge tube; (6) Transfer the cell supernatant from step (5) to an ultrafiltration centrifuge tube, centrifuge at 4000 g for 40 min, gently mix the supernatant concentrate in the cell concentration tank with a 200 μl pipette, pass the concentrate through a 0.22 μm syringe filter, and transfer it to a new ultrafiltration centrifuge tube using a 1 mL syringe; (7) Centrifuge at 100,000 g for 70 min at 4°C, discard the supernatant with a pipette, add 200 μl of sterile PBS, and slowly resuspend the precipitate to dissolve it; (8) Transfer the resuspended solution to a new ultrafiltration centrifuge tube, slowly add sterile PBS to the neck of the tube, centrifuge at 100000g for 70 minutes at 4°C, discard the supernatant, add 100ul sterile PBS to resuspend the precipitate, and finally obtain Schneider membrane mesenchymal stem cell-derived exosomes SMMSCs under non-osteoblastic induction conditions. exos , abbreviated as exos, placed at 4℃ for standby use and stored at -80℃ for long-term storage.

4. The method for preparing the nested microsphere hydrogel carrying dual exosomes according to claim 1, characterized in that: The specific process of step 2 includes: (1) 7.5 g of type A pigskin gelatin and 75 mL of sterile PBS were added to a conical flask containing 75 mL of sterile PBS in small amounts and several times. The mixture was placed in a 50°C water bath and stirred continuously for 1-2 h until the liquid in the conical flask became light yellow and clear. At this time, the type A pigskin collagen was completely dissolved, thereby obtaining a GelMA hydrogel precursor solution with a concentration of 10% w / v; (2) 750 μl of methacrylic anhydride was slowly added dropwise to the continuously stirred GelMA hydrogel precursor solution at a rate of 1 mL / min, and the reaction was continued for 3 h. (3) After 3 h, the pH of the GelMA hydrogel precursor solution was adjusted to neutral or near neutral. The GelMA hydrogel precursor solution after the reaction was stopped was transferred to a dialysis bag with a retention rate of 8000-14000. The GelMA hydrogel precursor solution was dialyzed in 50°C distilled water for 2 weeks. The distilled water was replaced every 4 h in the first week and every 8 h in the second week. (4) Centrifuge the dialyzed GelMA hydrogel precursor solution at 6000-7000 rpm for 5 min at room temperature using a filter membrane; (5) After centrifugation, the supernatant was aspirated into a 10 cm culture dish, cooled to a jelly-like state, and placed in a -80°C refrigerator overnight; (6) The pre-frozen GelMA hydrogel precursor solution was taken out from the -80°C refrigerator and placed in a freeze dryer for freeze drying for 48 h to obtain a white porous foam-like GelMA hydrogel precursor.

5. The method for preparing the nested microsphere hydrogel carrying dual exosomes according to claim 1, characterized in that: The specific process of step three includes: weighing 50mg of Fibrinogen and dissolving it in 1mL of sterile PBS containing exosome o-exos concentrate to obtain a 5% Fibrinogen hydrogel precursor solution loaded with exosomes, adding 2mg of photoinitiator, and performing photocrosslinking polymerization under 405nm ultraviolet light for 5min to obtain Fibrinogen o-exos Microsphere hydrogel.

6. The method for preparing the nested microsphere hydrogel carrying dual exosomes according to claim 1, characterized in that: The specific process of step 4 includes: weighing 100mg of GelMA hydrogel precursor and dissolving it in 1mL of sterile PBS containing exosome concentrate, and then mixing with 10mg of Fibrinogen o-exos The microsphere hydrogel is fully mixed to obtain GelMA exos -Fibrinogen o-exos The hydrogel precursor solution was added with 2 mg of photoinitiator and photocrosslinked and polymerized under 405 nm UV light for 5 min to obtain GelMA. exos -Fibrinogen o-exos Nested microsphere hydrogel.

7. A nested microsphere hydrogel carrying dual exosomes, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the double exosome-loaded nested microsphere hydrogel according to claim 7 in preparing an osteogenic material for the maxillary sinus region.

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