Bone organ constructed based on oxygen-releasing immune hydrogel microspheres and preparation method and application thereof

Abstract

The invention relates to a bone organ constructed based on oxygen-releasing immune hydrogel microspheres and a preparation method and application thereof. An oxygen release material, macrophages and bone marrow mesenchymal stem cells are loaded in the oxygen release immune hydrogel microspheres. And adding the oxygen-releasing immune hydrogel microspheres into a culture medium for culturing to obtain the bone organ constructed based on the oxygen-releasing immune hydrogel microspheres. The oxygen release material (CaO2-HAp) is entrapped in the hydrogel microspheres, so that continuous oxygen supply can be provided in a defect area, the low-oxygen environment is improved, the activity of osteoblasts is improved, osteogenic differentiation is promoted, macrophages are entrapped by the microspheres, the immune microenvironment is regulated and controlled, the release of proinflammatory cytokines is reduced, the osteogenic efficiency is improved, and bone defect repair is accelerated. The micro-fluidic technology is utilized to accurately control the size and shape of the microspheres, so that the microspheres can adapt to irregular bone defects, bone organs are formed through long-term in-vitro culture, and the biological activity and adaptability of the bone repair material are improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering technology, and in particular relates to a bone organoid constructed based on oxygen-releasing immune hydrogel microspheres, and a preparation method and application thereof. Background Art

[0002] Repair of bone defects has always been a major challenge in the field of orthopedics and regenerative medicine, especially for large or complex bone defects, and existing treatment methods still have many limitations. At present, the main methods for repairing bone defects include autologous bone transplantation, allogeneic bone transplantation, artificial bone implantation and tissue engineering bone repair. Among them, autologous bone transplantation is considered to be the "gold standard" due to its superior osteogenic activity and immune compatibility, but there are problems such as donor site damage and limited bone volume. Although allogeneic bone transplantation can partially make up for the donor limitation, there are risks of immune rejection, poor bone integration ability and infectious diseases. Artificial bone materials (such as calcium phosphate, hydroxyapatite, etc.) are widely used in bone repair, but due to the lack of biological activity and the degradation rate that is difficult to match the bone regeneration process, the osteogenesis effect is limited.

[0003] Existing bone tissue engineering materials are difficult to adapt to the geometric structure of complex bone defects. Bone defects vary in shape, and traditional bone repair materials (such as artificial bones or scaffolds) are difficult to accurately adapt to the defect shape due to their strong rigidity, which may lead to insufficient integration of implant materials and host tissues.

[0004] In recent years, the development of bone tissue engineering technology has provided new ideas for bone defect repair. Among them, hydrogel microspheres have become a research hotspot due to their excellent biocompatibility, controllable mechanical properties and drug delivery capabilities. Studies have used hydrogel microspheres to encapsulate osteoblasts or growth factors to promote bone tissue regeneration. However, current bone repair materials still face problems such as insufficient oxygen supply, lack of immune regulation ability, and difficulty in adapting to irregular bone defects. These factors seriously affect the osteogenesis efficiency and clinical transformation of the materials. Summary of the invention

[0005] Based on the current situation that existing bone tissue engineering materials are difficult to adapt to the geometric structure of complex bone defects, and the bone repair materials prepared from existing hydrogel microspheres still face problems such as insufficient oxygen supply, lack of immune regulation ability and difficulty in adapting to irregular bone defects, the present invention provides a bone organoid constructed based on oxygen-releasing immune hydrogel microspheres, and a preparation method and application thereof.

[0006] The purpose of the present invention can be achieved by the following technical solutions: The present invention first provides an oxygen-releasing immune hydrogel microsphere, wherein the oxygen-releasing immune hydrogel microsphere contains oxygen-releasing material, macrophages and bone marrow mesenchymal stem cells (BMSCs).

[0007] In one embodiment of the present invention, the oxygen-releasing material is CaO2-Hap nanoparticles, and Hap is hydroxyapatite.

[0008] In one embodiment of the present invention, the preparation method of the oxygen-releasing material CaO2-Hap nanoparticles is: 1) Prepared PBS solution, adding CaO2 nanoparticles into the PBS solution to obtain a mixed liquid; 2) The mixed liquid is stirred magnetically to react and generate hydroxyapatite (Ca 10 (PO4)6(OH)2, HAp); 3) Centrifuge to remove the supernatant to obtain precipitated particles, and dry them to obtain CaO2-HAp nanoparticles.

[0009] In one embodiment of the present invention, in step 1), the concentration of the PBS solution is 100mM-150mM, and the amount of CaO2 nanoparticles and PBS solution is 1g-10g:100-200ml.

[0010] In one embodiment of the present invention, in step 2), the magnetic stirring time is 8-16 hours.

[0011] In one embodiment of the present invention, in step 2), during the reaction, CaO2 reacts with water to generate Ca(OH)2, which then reacts with H3PO4 in PBS to generate hydroxyapatite (Ca 10 (PO4)6(OH)2, HAp).

[0012] In one embodiment of the present invention, in step 3), the drying condition is drying at 37° C. overnight.

[0013] In one embodiment of the present invention, the macrophages are selected from RAW264.7.

[0014] The present invention further provides a method for preparing the oxygen-releasing immune hydrogel microspheres, comprising the following steps: 1) Add photoinitiator, CaO2-Hap nanoparticles, BMSCs and macrophages to the silk fibroin (SilMA) hydrogel solution and mix well to obtain a mixed solution; 2) Microfluidic technology is used to prepare microspheres with the mixed solution obtained in step 1) as the aqueous phase and the paraffin oil solution as the oil phase at a flow rate ratio of 15:80 ml / min. The microspheres are cross-linked by ultraviolet light to form oxygen-releasing immune hydrogel microspheres with stable mechanical properties, controllable and sustained oxygen release, immunomodulatory and osteogenesis abilities.

[0015] In one embodiment of the present invention, in step 1), the amount of photoinitiator, CaO2-HAp nanoparticles, BMSCs and macrophages to silk hydrogel solution is as follows: 0.25 wt % of photoinitiator LAP, 100-500 μg of CaO2-HAp particles, (6-10)×10 6 BMSCs and (1.5-2.5) × 10 6 Macrophages.

[0016] In one embodiment of the present invention, in step 2), the oil phase is specifically a paraffin oil solution containing 5-10 wt % Span80.

[0017] In one embodiment of the present invention, in step 2), the conditions for cross-linking by ultraviolet light irradiation are: cross-linking by irradiating with 365 nm ultraviolet light for 15-20 seconds.

[0018] In one embodiment of the present invention, step 2) is followed by step 3): 3) Collect the microspheres prepared in step 2), centrifuge, remove the oil phase in the upper microspheres, then add PBS solution, mix and centrifuge again, repeat the washing 3-5 times, aspirate the upper liquid each time until the oil phase is completely removed to ensure that the microspheres are clean and remove any residual organic solvent, and finally obtain clean oxygen-releasing immune hydrogel microspheres.

[0019] In one embodiment of the present invention, in step 3), the centrifugation condition is: centrifugation at 500-1000 xg for 1-2 min.

[0020] The present invention further provides a method for preparing bone organoids constructed based on oxygen-releasing immune hydrogel microspheres, comprising the following steps: adding the oxygen-releasing immune hydrogel microspheres to a culture medium for culturing to obtain bone organoids constructed based on oxygen-releasing immune hydrogel microspheres.

[0021] In one embodiment of the present invention, the culture medium is DMEM, based on which 10% fetal bovine serum (FBS), 1% double antibody (penicillin-streptomycin), 10 mmol / L sodium β-glycerophosphate, 50 μg / mL ascorbic acid (Vc) and 10 nmol / L dexamethasone are added to promote osteogenic differentiation.

[0022] In one embodiment of the present invention, the culture conditions are as follows: the culture system is placed in a 37°C, 5% CO2 incubator for long-term culture, and the culture time is set to 28-30 days. During the culture process, the culture medium is replaced every 2-3 days to maintain the growth environment of the cells. From the 14th day onwards, the culture medium is adjusted to be replaced every day to ensure that the cells continue to obtain sufficient nutrition supply, optimize the osteogenic microenvironment, optimize the bone matrix deposition and mineralization process, and improve the osteogenic ability of the bone organoid unit.

[0023] During the culture process, morphological observations were performed: morphological changes of bone organoid units were observed regularly using an optical microscope, the thickening of microspheres, cell distribution status, and bone matrix deposition were recorded, and the growth and maturity of organoids were evaluated.

[0024] Functional verification was performed during the culture process: live-dead staining, ALP staining, ARS staining and Micro-CT analysis were performed on the bone organoid units at 28-30 days of culture to evaluate cell survival rate, osteogenic activity, mineralization capacity and bone matrix deposition, and to verify the functional characteristics of the bone organoid units constructed in vitro.

[0025] The present invention further provides bone organoids constructed based on oxygen-releasing immune hydrogel microspheres prepared based on the above preparation method.

[0026] The present invention further provides the use of bone organoids constructed based on oxygen-releasing immune hydrogel microspheres prepared based on the above preparation method in the preparation of bone defect repair materials.

[0027] After being cultured in vitro, bone organoid units can be used for personalized bone defect repair and are suitable for regenerative treatment of large-area bone defects.

[0028] In the process of obtaining the scheme of this application, the inventors found that insufficient oxygen supply is one of the key factors affecting the repair effect of bone tissue engineering materials. Due to the high vascularization of bone tissue, the survival and osteogenic differentiation of osteocytes depend on adequate oxygen supply. However, during the repair of bone defects, due to insufficient local vascularization, a hypoxic environment often results, thereby inhibiting the proliferation and differentiation of osteoblasts and inducing adverse inflammatory responses. Therefore, the present application encapsulates oxygen-releasing materials in hydrogel microspheres to provide a continuous oxygen supply in the defect area, improve the hypoxic environment, increase the activity of osteoblasts, and promote osteogenic differentiation.

[0029] The lack of immunoregulatory ability also limits the application of bone repair materials. Studies have shown that the bone repair process not only involves the proliferation and differentiation of osteoblasts, but is also significantly affected by the immune microenvironment. In particular, the polarization state of macrophages is crucial to bone regeneration, among which anti-inflammatory M2 macrophages can promote the differentiation of osteoblasts and reduce the inflammatory response by secreting anti-inflammatory factors. Therefore, the present application scheme combines immunoregulation, microspheres encapsulate macrophages, and regulate the immune microenvironment, reducing the release of proinflammatory cytokines, improving osteogenesis efficiency, and accelerating bone defect repair.

[0030] The principle of the present invention is described as follows: The present invention proposes a method for constructing bone organoid units based on oxygen-releasing immune hydrogel microspheres, which can further achieve efficient and accurate bone defect repair. The method uses microfluidic technology to prepare hydrogel microspheres and combines long-term in vitro culture strategies to optimize the formation process of bone organoids to improve bone regeneration ability.

[0031] The core of the present invention is to introduce a continuous oxygen release system and immune regulation function, so that the microspheres can improve the local microenvironment while promoting osteogenesis, thereby accelerating the bone repair process.

[0032] In the process of preparing microspheres, silk fibroin hydrogel (SilMA) was selected as the supporting material, and calcium peroxide-hydroxyapatite (CaO2-HAp) particles, bone marrow mesenchymal stem cells (BMSCs), and macrophages (RAW264.7) were encapsulated by microfluidic technology to achieve oxygen release, osteogenesis, and immune regulation. The design of oxygen release can effectively alleviate the common hypoxic environment in the process of bone defect repair, promote the survival and differentiation of osteoblasts, and the introduction of macrophages helps to optimize the local immune microenvironment, reduce inflammatory response and promote angiogenesis.

[0033] In addition, the present invention adopts a long-term in vitro culture strategy to construct a bone organoid unit with good osteogenesis ability. The growth of bone organoids was observed by optical microscopy, live-dead staining, ALP staining and ARS staining, and the bone matrix deposition was evaluated by Micro-CT analysis. The results show that the long-term cultured bone organoid units performed well in terms of cell survival rate, osteogenic activity and mineralization ability. In order to verify the effect of this method in repairing bone defects in vivo, the present invention further carried out a mouse skull defect model experiment. The bone organoid units cultured in vitro for 28 days were implanted into the mouse skull defect area. The results showed that the defect site was well repaired after the implantation of the bone organoid units, which further verified the role of bone organs in promoting bone regeneration and optimizing the immune microenvironment.

[0034] Compared with traditional bone repair strategies, the present invention has significant advantages. The continuous oxygen release system improves the local hypoxia problem, increases cell survival rate and osteogenesis efficiency; the immunomodulatory function optimizes the inflammatory response and promotes the immune balance in the bone tissue repair process; the bone organoid units constructed by long-term in vitro culture can adapt to the host environment more quickly after implantation, accelerating the repair process of bone tissue. Compared with traditional bone tissue engineering methods, the present invention has significant advantages in the accuracy of bone repair, osteogenesis efficiency and immunomodulatory ability, providing a new personalized treatment strategy for large-scale bone defect repair.

[0035] Compared with the prior art, the beneficial effects of the present invention are also reflected in the following aspects: The present invention achieves efficient cell survival and bone formation ability through long-term in vitro culture of bone organoids. The experimental results show that bone organoids exhibit high cell activity during the 28-day culture process. In addition, ALP and ARS staining results show excellent osteogenic differentiation and mineralization ability, proving that the present invention has the advantage of promoting bone matrix deposition and bone regeneration in long-term in vitro culture of bone organoids.

[0036] In the mouse skull defect repair experiment, the bone organoids used in the present invention significantly accelerated new bone formation after implantation. Micro-CT scan results showed that the experimental group formed more mature and continuous new bone tissue 8 weeks after surgery, while the bone defect area of ​​the control group still had a large number of unrepaired cavities, indicating that the bone organoids constructed by the present invention can effectively fill the defect area and promote the regeneration and repair of bone tissue.

[0037] The present invention uses oxygen-releasing immune hydrogel microspheres to simultaneously solve the problems of insufficient oxygen supply, immune regulation and conformability, and can achieve rapid repair of bone defects. The innovative points of the present invention include: ① Introducing an oxygen-releasing system: By encapsulating oxygen-releasing materials (such as CaO2-HAp) in hydrogel microspheres, a continuous oxygen supply can be provided in the defect area, the hypoxic environment can be improved, the activity of osteoblasts can be increased, and osteogenic differentiation can be promoted. ② Combined with immune regulation: The microspheres encapsulate macrophages and regulate the immune microenvironment, reduce the release of proinflammatory cytokines, improve osteogenesis efficiency, and accelerate bone defect repair. ③ Conformal bone organoid construction: Microfluidic technology is used to precisely control the size and morphology of the microspheres so that they can adapt to irregular bone defects. The obtained microspheres are uniformly spherical, with an adjustable diameter range of 150–800 μm. By adjusting the flow rate ratio of the water phase to the oil phase of the microfluidic device and the size of the droplet generation channel, the size of the microspheres can be precisely controlled. At the same time, the morphology of the microspheres can be adjusted by optimizing the silk fibroin concentration, cross-linker ratio and curing time to ensure that they have a stable structure during long-term in vitro culture and ultimately form bone organoids with bone formation and mineralization capabilities, thereby improving the bioactivity and adaptability of bone repair materials.

[0038] The solution of the present invention overcomes the defects of traditional bone repair materials and has broader clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 SEM image of CaO2-HAp; Figure 2 Electron microscopic image of oxygen-releasing immune hydrogel microspheres; Figure 3 Figure 28 of bone organoid cultured on day 28; Figure 4 Results of bone organoid functional evaluation; Figure 5 CT scan results of mouse skull defect. DETAILED DESCRIPTION

[0040] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0041] Example 1 The preparation of oxygen-releasing material CaO2-HAp is as follows: 1) Prepare 100 ml of PBS (100 mM) solution in a test tube and weigh 2 g of CaO2 nanoparticles into the PBS solution.

[0042] 2) The mixed liquid was stirred under a magnetic stirrer for 8 h.

[0043] 3) The supernatant was removed by centrifugation to obtain precipitated particles, which were dried overnight at 37°C to obtain CaO2-HAp nanoparticles. Figure 1 As shown, its microscopic morphology characteristics are demonstrated. It can be observed from the image that the obtained CaO2-HAp nanoparticles present a nanoflower-like structure. This nanoflower-like morphology not only increases the specific surface area of ​​the particles, which helps to improve the reactivity of the material, but also provides more biological interfaces, which is conducive to cell adhesion, growth and biomineralization processes. In addition, the pores and layered arrangement of the nanostructure may help to slowly release oxygen and maintain the oxygen supply capacity of the local microenvironment. Therefore, the SEM characterization results prove that the CaO2-HAp nanoparticles were successfully synthesized and have excellent morphological characteristics, which are suitable for further application in oxygen-releasing bone repair materials.

[0044] Example 2 The preparation of oxygen-releasing immune hydrogel microspheres comprises the following steps: 1) Preparation of oxygen-releasing immune silk hydrogel: Prepare 1 ml of 15% silk (SilMA) hydrogel solution, add 0.25% of photoinitiator LAP and stir until completely dissolved. Add 200 μg of CaO2-HAp particles, 8×10 6 BMSCs and 2×10 6 RAW264.7 cells were mixed evenly.

[0045] 2) Microfluidic technology was used, with the aqueous phase being a SilMA hydrogel solution containing CaO2-HAp particles, BMSCs and RAW264.7 cells, and the oil phase being a paraffin oil solution containing 8% Span80. Microspheres were prepared at a flow rate ratio of 15:80 ml / min between the aqueous phase and the oil phase. The microspheres were cross-linked by 365 nm UV light irradiation for 20 s to form oxygen-releasing immune hydrogel microspheres with stable mechanical properties and controllable release ability.

[0046] 3) Collect the prepared microspheres in a centrifuge tube and centrifuge at 500 x g for 1 min to remove the oil phase in the upper microspheres. Then add PBS solution, mix well and centrifuge again. Repeat the washing 3 times, absorbing the upper liquid each time until the oil phase is completely removed. This step ensures that the microspheres are clean and any residual organic solvent is removed. Finally, oxygen-releasing immune hydrogel microspheres are obtained. Figure 2 As shown in the figure, it can be observed that the obtained microspheres have a uniform spherical structure, consistent particle size, smooth surface and no obvious fragments, indicating that the microfluidic preparation process can accurately control the morphology and size of the microspheres. In addition, CaO2-HAp nanoparticles and cell components are uniformly distributed inside the microspheres. This structural feature helps to continuously release oxygen and provide cells with a suitable three-dimensional culture environment to support the proliferation and differentiation of osteoblasts. Overall, Figure 2 This shows that the microsphere system was successfully constructed and has good morphological uniformity and structural integrity, providing a material basis for subsequent applications in bone tissue engineering.

[0047] Example 3 A method for preparing bone organoids based on oxygen-releasing immune hydrogel microspheres comprises the following steps: 1) Cell culture: The oxygen-releasing immune hydrogel microspheres prepared in Example 2 were added to a culture medium, wherein the culture medium was DMEM supplemented with 10% fetal bovine serum (FBS), 1% double antibody (penicillin-streptomycin), 10 mmol / L sodium β-glycerophosphate, 50 μg / mL ascorbic acid (Vc) and 10 nmol / L dexamethasone to promote osteogenic differentiation.

[0048] 2) Culture conditions: The culture system was placed in a 37°C, 5% CO2 incubator for long-term culture, and the culture time was set to 28 days. During the culture process, the culture medium was replaced every 2 days to maintain the cell growth environment. From the 14th day onwards, the culture medium was changed every day to ensure that the cells continued to receive sufficient nutrition and optimize the osteogenic microenvironment.

[0049] 3) Morphological observation: Regularly use an optical microscope to observe the morphological changes of bone organoid units, record the thickening of microspheres, cell distribution and bone matrix deposition, and evaluate the growth and maturity of organoids. Figure 3 As shown, Figure 3 The images are optical microscope observation images of oxygen-releasing immune hydrogel microspheres at different time points (7 days, 14 days, 21 days, and 28 days) in vitro. The images were obtained by regularly collecting optical microscope photos of the bone organoid units in culture, and are mainly used to evaluate the morphological changes, cell distribution, and bone matrix deposition of the microspheres. Figure 3 It can be observed that as the culture time increases, the overall structure of the microspheres gradually thickens, and the cell density on the surface and inside gradually increases, indicating that the cells continue to proliferate in the microspheres. By day 14, the cells began to adhere extensively to the wall and gradually tended to aggregate. By day 21, the edges of the microspheres became clearer and the cell density increased significantly, suggesting that the cells had formed a stable three-dimensional tissue structure. By day 28, obvious bone matrix deposition appeared on the surface and inside of the microspheres, showing a darker color, reflecting the progress of the mineralization process, indicating that the bone organoid units have basically matured, providing a solid foundation for further in vivo transplantation.

[0050] 4) Functional verification: Live-dead staining, ALP staining, ARS staining and Micro-CT analysis were performed on the bone organoid units after 28 days of culture to evaluate cell survival rate, osteogenic activity, mineralization ability and bone matrix deposition, and verify the functional characteristics of the bone organoid units constructed in vitro (results refer to Figure 4 ). Figure 4 The experimental results of functional verification of bone organoid units after 28 days of in vitro culture, including live cell staining, dead cell staining, alkaline phosphatase (ALP) staining, Alizarin red (ARS) staining and Micro-CT analysis. These images were taken by optical microscopy and fluorescence microscopy, respectively, to evaluate cell survival rate, osteogenic activity, mineralization ability and bone matrix deposition, in order to verify the functional characteristics of bone organoid units constructed in vitro. Figure 4 The live and dead cell staining results show that the oxygen-releasing immune hydrogel microspheres still have a high cell survival rate (green fluorescence) after 28 days of culture, while the number of dead cells (red fluorescence) is relatively small, indicating that the microsphere material can maintain good cell survival during long-term culture. The ALP staining results showed that the ALP activity of osteoblasts in the microspheres was significantly enhanced, indicating that they have good osteogenic induction ability. ARS staining further verified that the microspheres can significantly promote mineralization after long-term culture, showing dark red mineralized nodules. In addition, the Micro-CT analysis results showed that dense bone matrix deposition was formed inside the bone organoid units, further proving that the system can effectively promote the formation of bone tissue. Figure 4 The experimental results showed that bone organoid units constructed with oxygen-releasing immune hydrogel microspheres could maintain high cell activity after long-term in vitro culture, promote osteogenic differentiation and mineralization, and form a microenvironment similar to natural bone tissue, confirming its application potential in bone tissue engineering and regenerative medicine.

[0051] Example 4 In vivo skull defect repair experiment, including the following steps: 1) Animal model construction: 4-5 week old C57BL / 6 mice were selected, and a critical size bone defect model with a diameter of 3 mm was prepared in the center of the skull using a high-speed drill under general anesthesia and aseptic conditions.

[0052] 2) Bone organoid implantation: Bone organoid units cultured in vitro for 28 days (obtained by the method of Example 3) were implanted into the defect site, cultured as a control group with no implantation, and the skin of the defect area was directly sutured. The health status of the mice was monitored postoperatively to ensure the stability and repeatability of the experiment.

[0053] 3) Tissue repair assessment: 8 weeks after surgery, mice were euthanized, skull samples were removed, and soft tissue residues were removed. The samples were then fixed in 4% paraformaldehyde solution for 24 hours, washed with deionized water, and dehydrated with graded ethanol. The dehydrated samples were used for Micro-CT scanning to evaluate the new bone formation in the bone defect area (results are shown in Figure 5 as shown). Figure 5 The three-dimensional reconstruction images obtained by Micro-CT scanning 8 weeks after surgery are shown to evaluate the effects of different treatment groups on the repair of skull defects. The left side is the control group (bone organoid units constructed without implantation of oxygen-releasing immune hydrogel microspheres), and the right side is the experimental group (implantation of bone organoid units). The blue area in the image represents the unrepaired defect area, while the white part represents the new bone tissue. Figure 5 It can be seen that the defect area in the control group still maintained a large cavity, and no significant new bone formation was observed, indicating that the group's own repair ability was limited. In the experimental group, a large amount of new bone formation was observed in the bone defect area, the bone volume in the defect area increased significantly, and a more uniform bone bridge connection structure was presented, indicating that the bone organoid unit constructed by oxygen-releasing immune hydrogel microspheres has significant advantages in promoting bone regeneration. This result proves that this strategy can effectively accelerate the repair process of large-sized bone defects, enhance bone regeneration ability, and is expected to be applied to personalized bone repair treatment.

[0054] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure 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. A method for preparing oxygen-releasing immune hydrogel microspheres, characterized in that: The following steps are involved: 1) Add photoinitiator, CaO2-Hap nanoparticles, BMSCs and macrophages to the silk hydrogel solution and mix well to obtain a mixed solution; 2) using microfluidic technology, the mixed solution obtained in step 1) is used as the aqueous phase, the paraffin oil solution is used as the oil phase, and the flow rate ratio of the aqueous phase to the oil phase is 15:80 ml / min to prepare microspheres, and the microspheres are cross-linked by ultraviolet light to form oxygen-releasing immune hydrogel microspheres with controllable sustained oxygen release, immunomodulatory ability and osteogenic ability; Among them, Hap is hydroxyapatite and BMSCs is bone marrow mesenchymal stem cells.

2. The method for preparing oxygen-releasing immune hydrogel microspheres according to claim 1, characterized in that: In step 1), the dosage of photoinitiator, CaO2-HAp nanoparticles, BMSCs and macrophages to silk hydrogel solution is as follows: 0.25 wt% photoinitiator LAP, 100-500 μg CaO2-HAp particles, (6-10)×10 6 BMSCs and (1.5-2.5) × 10 6 Macrophages.

3. The method for preparing oxygen-releasing immune hydrogel microspheres according to claim 1, characterized in that: After step 2), also perform step 3): 3) Collect the microspheres prepared in step 2), centrifuge, remove the oil phase in the upper microspheres, then add PBS solution, mix and centrifuge again, repeat the washing 3-5 times, aspirate the upper liquid each time until the oil phase is completely removed to ensure the microspheres are clean and remove the residual organic solvent, and finally obtain clean oxygen-releasing immune hydrogel microspheres.

4. An oxygen-releasing immune hydrogel microsphere, characterized in that: The oxygen-releasing immune hydrogel microspheres are prepared based on the preparation method according to any one of claims 1 to 3, wherein the oxygen-releasing material CaO2-Hap nanoparticles, macrophages and BMSCs are encapsulated in the oxygen-releasing immune hydrogel microspheres.

5. A method for preparing bone organoids based on oxygen-releasing immune hydrogel microspheres, characterized in that: The method comprises the following steps: adding the oxygen-releasing immune hydrogel microspheres according to claim 4 into a culture medium for culturing, and obtaining bone organoids constructed based on the oxygen-releasing immune hydrogel microspheres.

6. The method for preparing bone organoids based on oxygen-releasing immune hydrogel microspheres according to claim 5, characterized in that: The culture medium is DMEM, which is supplemented with 10% fetal bovine serum, 1% double-antibiotic penicillin-streptomycin, 10 mmol / L sodium β-glycerophosphate, 50 μg / mL ascorbic acid and 10 nmol / L dexamethasone. The culture conditions are as follows: the culture system is placed in a 37°C, 5% CO2 incubator for long-term culture for 28-30 days. During the culture process, the culture medium is replaced every 2-3 days. From the 14th day onwards, the culture medium is replaced every day.

7. A bone organoid constructed based on oxygen-releasing immune hydrogel microspheres, characterized in that: It is prepared based on the preparation method described in claim 5 or 6.

8. Use of the bone organoid constructed based on oxygen-releasing immune hydrogel microspheres as claimed in claim 7 in the preparation of bone defect repair materials.

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