Injectable mineralized hydrogel microspheres as well as preparation method and application thereof

Abstract

The invention provides injectable mineralized hydrogel microspheres as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. According to the invention, methacrylic anhydride gelatin, methacrylic anhydride sodium alginate and bone cell membrane vesicle blended hydrogel microspheres are prepared through gas shearing microfluidic and photopolymerization technologies, and mineralized hydrogel microspheres are prepared through in-situ pre-mineralization. The compression modulus of the mineralized hydrogel microspheres is obviously enhanced, and amorphous calcium phosphate particles are formed in the gel fibers in situ. The mineralized hydrogel microspheres can effectively promote osteogenic differentiation of BMSCs, and cells adhered to the surfaces of the microspheres can express bone cell markers more quickly. In addition, the material has good blood vessel formation regulating and controlling capacity, formation of a mature bone cell network can be accelerated in an in-vivo environment, and bone defect healing is accelerated. Therefore, the mineralized hydrogel microspheres can promote intelligent bone regeneration by accelerating bone cell network reconstruction.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to an injectable mineralized hydrogel microsphere and a preparation method and application thereof. Background Art

[0002] At present, bone regeneration of critical size is still a challenging problem, among which the massive loss of osteocytes is one of the important reasons for disordered bone regeneration. In the process of bone repair, osteocytes are like "battlefield commanders" who can respond quickly to mechanical and biological signals, and play a commanding function through the complex network formed by cytoplasmic extension, precisely regulating the coordination and unity between osteogenesis, osteoclasts and microvascular formation. However, osteocytes in critical bone defect sites are severely lost, and autologous seed cells (such as periosteal stem cells, bone marrow mesenchymal stem cells, etc.) are difficult to quickly transform into osteocytes and form a regulatory network. Therefore, it is necessary to find more effective strategies to enhance osteocyte network reconstruction. Rapid reconstruction of functional osteocyte networks requires the combined action of exogenous environmental factors and endogenous biological signals. Specifically, osteocytes are formed by osteoblasts being embedded in autocrine mineralized matrix. The mineralized environment outside the cell is the basis for osteocyte formation, and biological signals are molecular switches that determine the fate of cell differentiation. Therefore, by providing a suitable environment for osteocyte differentiation and combining precise signal pathway regulation, it is expected to achieve rapid osteocyte network reconstruction.

[0003] A good mineralization environment is the basis for the formation of bone cell networks. Previous studies have tried various methods to provide a mineralization environment, including calcium phosphate bone cement, and have achieved good therapeutic effects. However, simply stacking calcium ions and phosphates is unscientific, and exogenously supplemented minerals need to be degraded before they can participate in bone reconstruction. More and more studies have shown that amorphous calcium phosphate (ACP) can be directly acquired by osteoblasts and integrated into the bone matrix. In situ mineralization technology can dynamically mineralize in the three-dimensional network of hydrogels, thereby forming amorphous minerals in situ, which provides ideas for achieving an ideal mineralization environment for bone cell network reconstruction.

[0004] Gulden Camci-Unal et al. reported that the simulated in situ mineralization process can form amorphous calcium phosphate particles in situ on hydrogel fibers. However, after mineralization, the hydrogel is in large blocks and cannot be minimally invasively injected. At the same time, its insufficient internal permeability leads to uneven mineralization and low cell adhesion rate. Therefore, how to use micron-sized hydrogel microspheres for mineralization treatment to form mineralized hydrogel microspheres that are more conducive to minimally invasive injection and cell adhesion has become a new idea.

[0005] In addition, matrix hardness is also an important environmental factor required for the formation of bone cell networks. Li et al. found that cells are more likely to differentiate into bone cells on a matrix with higher hardness. Interestingly, in situ premineralization can significantly increase the strength of the hydrogel, and 30 minutes of premineralization can increase the compression modulus by nearly 20 times. Therefore, providing an ideal mineralization environment and matrix hardness by premineralizing hydrogel microspheres is an effective way to accelerate the reconstruction of bone cell networks.

[0006] Osteocytes are the final form of osteoblast differentiation. Endogenous pathway signals are deeply involved in regulating the dramatic changes in morphology and function during osteoblast differentiation. Among them, spatial activation of the Notch pathway is the key to determining the fate of osteoblast differentiation. The Notch signal transmits the signal to the cell nucleus through three protease cleavages to regulate downstream gene expression. After the Notch pathway is activated during osteoblast differentiation, it will significantly promote cell mineralization, thereby accelerating osteoblast transformation and osteoblast network formation. The activation of the Notch pathway in osteocytes can stimulate the release of vascular growth factors and promote the formation of functional blood vessels (such as H-type blood vessels), which is also one of the ways in which the osteocyte network plays a commanding role. However, it is difficult to produce sufficient endogenous Notch signals in bone repair. At the same time, the results of Notch signal transduction are variable, and its activation results are highly dependent on the type of ligand and the binding form.

[0007] Komatsu et al. found (PMID: 38402584) that spatially bound Jagged1 (one of the main ligands of Notch) can participate in guiding the branching and propagation of the vascular tree. Bian et al. reported that short peptides that mimic Jagged1 can significantly promote bone repair and enhance cell mineralization ability. Therefore, it is a good choice to activate the Notch pathway through spatial Jagged1 ligands. However, Jagged1 is a transmembrane protein and must bind to the ligand in a membrane-bound form. Cell membrane vesicles can retain the cell membrane structure to the greatest extent and have more stable membrane protein activity, which provides a way to achieve spatial activation of the Notch pathway. By constructing bone cell membrane vesicles with high expression of Jagged1 and loading them in three-dimensional mineralized hydrogel microspheres, simulating spatial Jagged1 biological signals may be an effective way to accelerate the reconstruction of bone cell networks and the regulation of vascular regeneration.

[0008] However, existing mineralization methods often form mineralized substances on the surface or in the pores of hydrogels. This mineralization method will make the matrix of large hydrogels hard after mineralization, resulting in the difficulty of being non-injectable. On the other hand, due to the poor stability of the existing hydrogel network, the stability of mineralized hydrogels needs to be further improved. At the same time, existing mineralized hydrogels cannot form amorphous calcium phosphate in situ in the hydrogel, which makes the effect of bone cell regeneration extremely effective.

[0009] Therefore, how to provide an injectable mineralized hydrogel microsphere while significantly improving the stability of the mineralized hydrogel and forming amorphous calcium phosphate to significantly promote the regeneration of bone cells has become a technical problem that needs to be urgently solved in the field of bone repair. Summary of the invention

[0010] The present invention is to solve the above technical problems, thereby providing an injectable mineralized hydrogel microsphere and its preparation method and application. The technical purpose of the present invention is to solve the problem that the existing mineralized hydrogel will form a large hard matrix after mineralization and thus cannot be injected, and provide an injectable mineralized hydrogel microsphere; on the other hand, solve the problem that the existing hydrogel network has poor stability, the material stability after mineralization is poor, and the amorphous calcium phosphate cannot be formed in situ, and provide a mineralized hydrogel microsphere with good structural stability, which can form amorphous calcium phosphate in situ and significantly promote bone regeneration.

[0011] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0012] The present invention first provides a method for preparing injectable mineralized hydrogel microspheres, comprising the following steps:

[0013] (1) Cultivating bone cells in a basal medium to prepare a recombinant adenovirus, wherein the adenovirus vector contains coding regions for Jagged1, shJagged1, red fluorescent protein, and green fluorescent protein; then transfecting the bone cells with the adenovirus, collecting the total RNA after transfection, preparing cDNA by reverse transcription, collecting the bone cells, and preparing osteoblast membrane vesicles, which are recorded as Jag1-OMVs;

[0014] (2) preparing methacrylic anhydride gelatin and methacrylic anhydride sodium alginate hydrogels, and then forming an aqueous phase solution with methacrylic anhydride gelatin, methacrylic anhydride sodium alginate, Jag1-OMVs and a photosensitizer, using nitrogen as the gas phase, and preparing hybrid hydrogel microspheres through a gas shear microfluidic device;

[0015] (3) Using calcium chloride solution to collect microdroplets, and simultaneously using blue light for cross-linking, the collected hydrogel microspheres are subjected to gradient mineralization to obtain the injectable mineralized hydrogel microspheres.

[0016] The present invention first prepares osteocyte membrane vesicles with high expression of Jagged1 by nano-extrusion method, and then uses gas flow control technology and in situ mineralization technology to prepare mineralized hydrogel microspheres blended with GelMA and AlgMA. The present invention successfully constructs a mineralized hydrogel microsphere loaded with osteocyte membrane vesicles by combining microenvironment induction and biological signal regulation, which can accelerate the reconstruction of osteocyte network and promote intelligent bone regeneration. The experimental results show that the compression modulus of the mineralized hydrogel microsphere is significantly enhanced, and uniformly distributed amorphous calcium phosphate particles are observed in the gap of the gel matrix. In vitro experiments have shown that the mineralized hydrogel microsphere has good biocompatibility, and the induced BMSCs highly express osteocyte markers and Notch pathway markers. Further co-culture experiments found that it has obvious ability to regulate angiogenesis in vitro. Ectopic bone regeneration experiments suggest that mineralized hydrogel microspheres can quickly form osteocyte network structures in vivo. Subsequently, animal experiments suggest that mineralized hydrogel microspheres can significantly promote bone repair of critical bone defects. Therefore, the mineralized hydrogel microspheres provided by the present invention promote intelligent bone regeneration by accelerating the reconstruction of bone cell networks, opening up a new treatment method for the treatment of clinical bone injuries in the future.

[0017] Compared with the existing mineralized hydrogels, the mineralized hydrogel microspheres prepared by the present invention avoid the difficulty of existing bulk hydrogels becoming hard and non-injectable after mineralization. At the same time, the present invention uses a double cross-linked hydrogel network to significantly improve the stability of the hydrogel, so that it can maintain a stable structure in a phosphoric acid solution. At the same time, because the present invention first uses Ca 2+ Ionic crosslinking allows calcium phosphate to be formed at the intersection of the hydrogel network rather than in the hydrogel pores, and this method significantly improves the firmness of the combination of calcium phosphate and hydrogel. However, as shown in the preparation method of the comparative example of the present invention, by first preparing hydrogel microspheres and then performing gradient mineralization, mineralized substances cannot be formed at the intersection of the hydrogel network, but mineralized substances are formed in the hydrogel pores. This method cannot form amorphous calcium phosphate, thereby reducing the regeneration effect of mineralized hydrogel microspheres on bone defects.

[0018] Furthermore, the bone cells in step (1) are cell line MLO-Y4, and the basal culture medium is a high-glucose DMEM culture medium containing 10 wt % fetal bovine serum.

[0019] Furthermore, in step (1), the bone cells are transfected with adenovirus for 24 hours, and the total RNA is collected 48 hours after transfection.

[0020] Furthermore, the preparation method of the methacrylic anhydride gelatin in step (2) is: dissolving gelatin in water, adding methacrylic acid dropwise at 4°C in the dark, mixing evenly, then adding NaOH solution, putting the obtained liquid into a dialysis bag, and lyophilizing after dialysis to obtain methacrylic anhydride gelatin; the preparation method of the methacrylic anhydride sodium alginate is: dissolving sodium alginate in water, adding methacrylic acid dropwise at 4°C in the dark, mixing evenly, then adding NaOH solution, putting the obtained liquid into a dialysis bag, and lyophilizing after dialysis to obtain methacrylic anhydride sodium alginate.

[0021] Furthermore, the weight ratio of methacrylic anhydride gelatin, methacrylic anhydride sodium alginate, Jag1-OMVs and photosensitizer in the aqueous solution in step (2) is 4:0.5:0.1:0.5.

[0022] Furthermore, the flow rate of the nitrogen in step (2) is 1 L / min.

[0023] Furthermore, in step (3), blue light cross-linking is performed for 15 minutes, and the concentration of the sodium phosphate dihydrate solution is 200 mmol / L.

[0024] Furthermore, the gradient mineralization process in step (3) is to oscillate and soak in a sodium phosphate dihydrate solution for 30 minutes, then transfer to a calcium chloride solution and continue oscillating and soaking for 30 minutes, and complete the gradient mineralization process of the hydrogel microspheres by repeated soaking for 24 hours.

[0025] The second object of the present invention is to provide injectable mineralized hydrogel microspheres prepared by the method as described above.

[0026] The third object of the present invention is to provide the use of the injectable mineralized hydrogel microspheres as described above in the preparation of drugs for treating bone defect regeneration.

[0027] The beneficial effects of the present invention are as follows:

[0028] The present invention constructs an injectable mineralized hydrogel microsphere, which can provide a mineralized environment and matrix hardness suitable for the reconstruction of bone cell networks. At the same time, Notch signaling is activated through the osteocyte membrane vesicle space with high expression of Jagged1, promoting osteocyte differentiation and enhancing its vascular regulation function. The results show that the compression modulus of the mineralized hydrogel microsphere is significantly enhanced, and amorphous calcium phosphate particles are formed in situ in the gel fibers. In vitro experiments have shown that the mineralized hydrogel microspheres effectively promote the osteogenic differentiation of BMSCs, and the cells adhering to the surface of the microspheres can express osteocyte markers more rapidly. Subsequent co-culture experiments have found that it has a good ability to regulate angiogenesis. Ectopic bone regeneration experiments suggest that mineralized hydrogel microspheres can accelerate the formation of mature osteocyte networks in the in vivo environment. Subsequent critical bone defect experiments of the femur suggest that mineralized hydrogel microspheres can accelerate bone defect healing. In short, this mineralized hydrogel microsphere promotes intelligent bone regeneration by accelerating the reconstruction of the osteocyte network, providing a promising new solution for the treatment of critical bone defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of mineralized hydrogel microspheres loaded with Jag1-OMVs promoting bone cell transformation and activation; (A) Flow chart of osteocyte membrane vesicle preparation; (B) Preparation process of Jag1-OMVs-loaded mineralized hydrogel microspheres; (C) Application of mineralized hydrogel microspheres in rat femoral defect model and its mechanism of action in promoting bone cell transformation and activation.

[0030] Figure 2 Morphological characterization and biological function of Jag1-OMVs; (A) Flow chart of preparation of osteocyte membrane vesicles; (B) Adenovirus transfection of MLO-Y4 cells; (C) PCR verification of changes in Jagged1 gene expression in osteocytes; (D) Typical transmission electron microscopy images of OMVs; (E) Nanoparticle tracking analysis of OMVs particle size; (F) Western blotting to verify the protein distribution of CD63 (vesicle marker), CD81 (vesicle marker), Jagged1 (surface protein) and NakATPase (membrane protein internal reference) in OMVs; (G, H) Cell immunofluorescence staining to detect Notch1, VEGFA (G) and DLL1, Runx2 (H); (I) PCR verification of the ability to regulate angiogenesis (*p<0.05, **p<0.01; data are expressed as mean ± standard deviation, n=3).

[0031] Figure 3Characterization of the morphology of mineralized hydrogel microspheres; (A) Preparation process of mineralized hydrogel microspheres (MHMs); (B) Micromorphology of MHMs; (C) Loading effect of Dio fluorescently labeled Jag1-OMVs; (D) Uniformity and particle size distribution of MHMs; (E) Scanning electron microscopy (SEM) observation of the morphology of hydrogel microspheres (HMs) and element distribution; (F) Quantitative element distribution spectrum of MHMs; (G) Morphology and element distribution of MHMs, the red arrow indicates the in situ formation of calcium phosphate; (H) Quantitative element distribution spectrum of MHMs; (I) Changes in rheological properties of MHMs; (J) Thermogravimetric analysis of MHMs; (K) Fourier transform infrared spectroscopy (FTIR) results of MHMs; (L) X-ray diffraction (XRD) analysis of MHMs.

[0032] Figure 4 Biocompatibility and osteoblast transformation ability of mineralized hydrogel microspheres; (A) Live / dead cell staining to evaluate the cytotoxicity of BMSCs (bone marrow mesenchymal stem cells) and HUVECs (human umbilical vein endothelial cells); (B) i: osteoblast transformation experimental process; ii: co-culture experimental steps; (C) Immunofluorescence staining of cells in mineralized hydrogel microspheres; (D) DMP1 (dentine matrix protein 1) fluorescence quantitative analysis; (E) PCR detection results of osteoblast-related genes (*p<0.05, **p<0.01; data are expressed as mean ± standard deviation, n=3). Figure 5 Figure 3 The ability of mineralized hydrogel microspheres to promote osteodifferentiation and activate angiogenesis; (A) ALP (alkaline phosphatase) staining to evaluate early osteogenic activity; (B) Alizarin red staining to detect late osteogenic ability; (C) PCR analysis of the expression levels of genes related to osteogenic differentiation; (D) angiogenesis assay (tube formation assay); (E) HUVECs (human umbilical vein endothelial cells) scratch assay (cell migration); (F) Quantitative data of tube formation; (G) Quantitative analysis of scratch healing rate (*p<0.05, **p<0.01; data are expressed as mean ± SD, n=3).

[0033] Figure 6 To evaluate the regenerative potential of osteoblast networks of mineralized hydrogel microspheres in a low-inflammatory environment; (A) Flowchart of the ectopic osteogenesis experimental method; (B) Visualization of microvascular structure in bone grafts and MicroCT three-dimensional reconstruction; (C) Quantitative data of MicroCT analysis; (D) Histological examination of paraffin sections with H&E and Masson staining; (E) Immunofluorescence staining results and (F) quantitative analysis of fluorescence data (*p<0.05, **p<0.01; data are expressed as mean±SD, n=5).

[0034] Figure 7Image evaluation of the critical bone defect model of SD rats treated with mineralized hydrogel microspheres; (A) Establishment of the femoral defect model in rats; (B) Micro-CT three-dimensional reconstruction image of the femur, showing the in vivo regeneration effect; (C) Three-dimensional reconstruction of the bone defect area observed by Micro-CT; (D) Microstructural parameters of the new bone tissue, including bone mineral density (BMD), bone volume / total volume (BV / TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N), (*p<0.05, **p<0.01; data are expressed as mean ± SD, n=5).

[0035] Figure 8 Histological evaluation of the critical bone defect model of SD rats treated with mineralized hydrogel microspheres; (A) Hematoxylin-eosin (H&E) and Masson's trichrome (Masson) staining; (B, C, D, E) bone histopathological scores (*p<0.05, **p<0.01; data are expressed as mean ± SD, n=5).

[0036] Fig. 9 Immunofluorescence evaluation of the critical bone defect model of SD rats treated with mineralized hydrogel microspheres; (A, C) OPN (osteopontin) and OCN (osteocalcin) fluorescence staining and quantitative analysis; (B, C) CD31 (endothelial cell marker) and EMCN (endothelial cell mucin) fluorescence staining and quantitative analysis; (*p<0.05, **p<0.01; data are expressed as mean ± standard deviation, n=5).

[0037] Fig.10 For GelMA and AlgMA 1 HNMR results.

[0038] Fig.11 (A) Immunofluorescence staining of Notch1 and VEGFA; (B, C) and quantitative analysis of immunofluorescence intensity; (*p<0.05, **p<0.01; n=3 independent experiments, t-test and one-way ANOVA were used). DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is described in detail below in conjunction with the embodiments. It is necessary to point out that the following embodiments are only used to explain and illustrate the present invention and are not used to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still belong to the protection scope of the present invention.

[0040] Example 1

[0041] 1. Experimental methods and characterization

[0042] 1. Preparation and characterization of Jag1-OMVs

[0043] Preparation of Jag1-OMVs: First, the osteoblast cell line MLO-Y4 was purchased from the cell bank of the Chinese Academy of Sciences and cultured in high-glucose DMEM with 10% FBS. Subsequently, the recombinant adenovirus was prepared by AdEasy technology. The adenovirus vector contained the coding regions of Jagged1, shJagged1, red fluorescent protein (RFP), and green fluorescent protein (GFP). The titer was determined to be 1×10 8 MLO-Y4 cells were transfected with adenovirus at TU / mL for 24 h, and then the cell culture medium was replaced and cultured for another 24 h before observing the expression level of fluorescent protein under a fluorescence microscope. Total RNA was collected 48 h after transfection, and cDNA was prepared using a reverse transcription kit (TAKARA, Japan), followed by RT-PCR using SYBR Green. All primers used in the experiment are shown in Table 1.

[0044] Table 1 Primer sequences

[0045]

[0046] The preparation method of cell membrane vesicles is as follows: Use a cell scraper to scrape an appropriate amount of MLO-Y4 cells and centrifuge to obtain a precipitate. Use a membrane protein extraction kit (Beyond Sky, China), add membrane protein extraction buffer A, and repeatedly blow and beat in an ice bath for 15 minutes. After sufficient lysis, centrifuge at 4°C, 700g for 10 minutes, collect the supernatant, and then centrifuge the supernatant again at 12000g, 4°C for 30 minutes. The precipitate is the osteocyte cell membrane. The collected cell membranes pass through 0.8, 0.4, and 0.1μm polycarbonate membranes in turn, which are engineered cell membrane vesicles, which are collected and stored at 4°C.

[0047] Characterization of Jag1-OMVs: Morphology and size analysis were performed using transmission electron microscopy (TEM, Hitachi, HT7800) and NTA (ZetaView). Then, membrane proteins CD63 (Abcam, USA), CD81 (Abcam, USA), Jagged1 (Abcam, USA) and Na + / K + ATPase (Abcam, USA). Briefly, the cell membrane vesicles were centrifuged and precipitated, then treated with protein lysis buffer, added with sample buffer and fixed by boiling. Then gel electrophoresis, transfer, blocking, primary antibody incubation, secondary antibody incubation and development using Bio-Rad developer were performed.

[0048] 2. Functional identification of Jag1-OMVs

[0049] To verify the biological function of Jag1-OMVs, osteocytes were treated with cell membrane vesicles and the activation status of the Notch pathway was observed. The experimental groups were: OMVs, Jag1-OMVs and shJag1-OMVs.

[0050] Immunofluorescence was used to detect protein expression. After 24 h of treatment, cells were fixed with paraformaldehyde, washed thoroughly, and incubated overnight with anti-Notch1 (Abcam, USA), anti-VEGFA (Abcam, USA) antibodies, anti-Runx2 (Abcam, USA) and DLL1 (Abcam, USA). Fluorescent secondary antibodies were incubated at room temperature for 60 minutes, cell nuclei were stained with DAPI, cells were observed under a fluorescence microscope, and fluorescence quantification was performed using ImageJ software. RT-PCR was then used to detect changes in RNA expression, as described above.

[0051] 3. Preparation and characterization of mineralized hydrogel microspheres

[0052] The synthesis method of GelMA and AlgMA is as follows: 10g gelatin (Gel) or sodium alginate (Alg) is dissolved in 1000ml ultrapure water, and 20ml methacrylic acid (MA, Sigma-Aldrich, USA) is added dropwise at 4°C in the dark, and mixed evenly. Finally, NaOH solution (20mL, 5M) is added. The obtained liquid is placed in a dialysis bag, dialyzed for 3 days, and then freeze-dried to obtain the final product. GelMA and AlgMA are prepared by 1 The products were characterized by H NMR (600 MHz, Bruker, Germany).

[0053] Subsequently, a mixed hydrogel microsphere was prepared by a gas shear microfluidic device. The aqueous solution: 4wt% GelMA, 0.5wt% AlgMA, 0.1wt% Jag1-OMVs and 0.5wt% photosensitizer. Gas phase: nitrogen with a flow rate of 1L / min. The liquid phase caliber of the microfluidic device is 30G, and the gas phase caliber is 18G. Calcium chloride solution is used to collect microdroplets, and blue light is used for cross-linking for 15 minutes. The collected hydrogel microspheres are subjected to gradient mineralization, specifically in Na 3 PO 4 After 30 min of shaking and soaking in a sodium phosphate dihydrate solution (200 mmol / L), the hydrogel microspheres were transferred to a calcium chloride solution and continued to be shaken and soaked for 30 min. The gradient mineralization process of the hydrogel microspheres was completed by repeated soaking for 24 h. Finally, the microsphere morphology was observed by optical microscopy and scanning electron microscopy (FEI Sirion 200, USA), and the distribution of Dio-labeled Jag1-OMVs in the microspheres was observed by laser confocal microscopy (LSM800, Zeiss, Germany).

[0054] 4. Mechanical analysis of mineralized hydrogel microspheres

[0055] The mineralized hydrogel microspheres were tested for compression modulus using a rheometer (TA, USA). The mineralized hydrogel microspheres were spread flat, dried, and calibrated. The stress and strain of the samples were then tested under a controlled force of 0.1 N min-1 (maximum 2 N), and each set of experiments was repeated three times.

[0056] 5. Thermogravimetric analysis of mineralized hydrogel microspheres

[0057] Thermogravimetric experiments were used to analyze the weight proportion of minerals in the mineralized hydrogel microspheres. The freeze-dried mineralized hydrogel microspheres were placed in a Discovery TGA 550 instrument (TA, USA), and the heating rate was set to 10 k / min and heated to 800 °C. Each group of experiments was repeated three times.

[0058] 6. Composition analysis of mineralized hydrogel microspheres

[0059] Fourier transform infrared spectroscopy (FTIR) was used to characterize the mineralized hydrogel microspheres. The FTIR spectrometer (ThermoFisher Scientific, USA) was used to record the wavelength range from 500 to 4000 cm -1 FTIR spectrum. X-ray diffraction (XRD) was used to characterize the composition of mineralized hydrogel microspheres. The mineralized hydrogel microspheres were measured using an X-ray diffractometer (Rigaku Ultima IV, Japan) at a temperature of 5-90° and 5° / min.

[0060] 7. Cell biocompatibility of mineralized hydrogel microspheres

[0061] The cytotoxicity of mineralized hydrogel microspheres on BMSCs and HUVECs was evaluated by live-dead staining and cell counting kit-8 (CCK8) assays. The experimental groups included Control, HMs, MHMs, OMVs@MHMs, and Jag1-OMVs@MHMs. Specifically, in the live-dead staining assay, cells were co-incubated with microspheres for 3 days and stained using the Calcein–AM / PI kit. In the CCK8 assay, CCK-8 solution (Beyotime, China) was added to each group, and after 2 hours of reaction at 37°C in the dark, the absorbance of the solution was measured at 450 nm using a microplate reader (Molecular Devices, FlexStation 3).

[0062] 8. Identification of the osteogenic function of mineralized hydrogel microspheres

[0063] To identify the osteogenic function of mineralized hydrogel microspheres, we cultured BMSCs in osteogenic induction medium (10 mM glycerophosphate, 100 nM dexamethasone and 50 mg / ml ascorbic acid) and detected them by ALP staining and Alizarin red staining after 7 and 14 days of induction, respectively. The ALP staining and Alizarin red staining methods were described in previous studies. []RT-PCR was used to detect the expression of osteogenic-related genes in BMSCs treated with mineralized hydrogel microspheres (osteogenic differentiation genes, Runx2, Ost, OCN, Col-1 and OPN). The specific method is described in 1 above.

[0064] 9. Functional identification of mineralized hydrogel microspheres in promoting osteocyte differentiation and activation

[0065] To identify the ability of mineralized hydrogel microspheres to promote osteocyte differentiation and activation, we seeded BMSCs on the surface of the microspheres and continued to culture them in osteogenic conditioned medium for seven days, and then detected the expression levels of osteocyte markers by immunofluorescence staining and RT-PCR. The methods of immunofluorescence staining and RT-PCR were as described above, and the antibodies used in this experiment included: DMP1 (Abcam, United States), Notch1 (Affinity) and VEGFA (Abcam, United States).

[0066] To identify the ability of activated osteocytes to regulate angiogenesis in vitro, we used a co-culture system, placed microspheres in a transwell chamber, and performed cell scratch tests and angiogenesis tests in the lower layer. In the angiogenesis experiment, Matrigel was evenly spread on the bottom of the dish using a pre-cooled pipette tip and culture dish, and solidified in a 37°C incubator. After sufficient solidification, HUVECs cells were spread, and the formation of tubules was observed after 6 hours. The image of the entire well was scanned and recorded using a fluorescence microscope, and then the tubule formation parameters were analyzed using ImageJ software. In the scratch experiment, when the density of HUVECs reached 80%, a cross wound scratch was formed with a 200μL pipette tip. The cross scratch was conducive to the accuracy of the position during observation, and the number of migrating cells and their migration speed were continuously observed.

[0067] 10. In vivo ectopic osteogenesis experiment of mineralized hydrogel microspheres

[0068] The in vivo ectopic osteogenesis experiment was performed as previously reported. Briefly, BMSCs were seeded on mineralized hydrogel microspheres and cultured for osteogenic induction in vitro for 5 days. Subsequently, the cell / microsphere mixture was subcutaneously injected into the flanks of nude mice (3 mice per group, 6-week-old females). The experimental groups included: Control group, HMs group, MHMs group, OMVs@MHMs group, and Jag1-OMVs@MHMs group. The control group was injected with an equal volume of PBS, which was completely absorbed 2 days after injection. At 4 weeks, the animals were euthanized to harvest the ectopic bone blocks. Micro-CT scanning was performed on the obtained bone blocks. After decalcification, they were embedded in paraffin and stained with hematoxylin-eosin (H&E), Masson staining, and immunofluorescence. The antibodies used for immunofluorescence included: OPN (Abcam, USA), Runx2 (Abcam, USA), CD31 (Affinity), and VEGFA (Abcam, USA). For specific experimental steps, see 11.

[0069] 11. Experimental study on implantation of mineralized hydrogel microspheres into large bone defects in SD rats

[0070] The animal experiments involved in this embodiment have been approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (approval number: IACUC-CQMU-2023-0108). Male Sprague-Dawley (SD) rats weighing between 320-280 g were randomly divided into 5 groups: Control group (n=10), HMs group (n=10), MHMs group (n=10), OMVs@MHMs group (n=10) and Jag1-OMVs@MHMs group (n=10). After anesthesia with 3% sodium pentobarbital (40 mg / kg), the femur was fully exposed, and a circular defect of about 1 mm deep and about 3.5 mm in size was drilled on the medial femoral neck with an electric drill. The corresponding biomaterials were injected at the defect, and the wound was sutured in layers.

[0071] Bone tissue specimens were obtained at 4 and 8 weeks after surgery and scanned using Micro-CT, including three-dimensional reconstruction, bone tissue volume / total tissue volume, trabecular thickness, bone density, and trabecular number. The bone tissue specimens were then decalcified, paraffin-embedded, and sliced. The sections were further dewaxed and hydrated and stained with hematoxylin-eosin (H&E) and Masson staining. Two assessors used the Mankin scoring system to evaluate bone healing. The specific steps of immunofluorescence staining were as described above. Briefly, the sections were permeabilized in 0.1% Triton X-100 for 15 minutes, then blocked in 5% goat serum for 30 minutes, and then incubated overnight with primary antibodies four times, including OPN (Abcam, USA), OCN (Abcam, USA), CD31 (Affinity), and Emcn (Abcam, USA). Fluorescent secondary antibodies were incubated at room temperature in the dark, DAPI was incubated for 5 minutes, and anti-fluorescence quencher was added and the sections were sealed before imaging using a fluorescence microscope.

[0072] 12. Statistical analysis

[0073] Statistical analysis was performed using GraphPad Prism 8.0 and Oringin. The statistical significance of the results was determined by Student's t test and one-way analysis of variance (ANOVA). Each experiment was repeated at least three times, and the results are expressed as mean ± standard deviation (*p<0.05, **p<0.01).

[0074] 2. Experimental Results and Discussion

[0075] 1. Characterization and functional identification of JAG1-OMVs osteocyte vesicles

[0076] In order to maximize the regulatory effect of activating osteocytes, it is necessary to deliver highly bioactive Jagged1 protein ligands. However, Jagged1 is a protein with multiple transmembrane structures, and it is difficult to exert normal biological functions after purification. Engineered cell membrane vesicles (mEVs) have a similar morphology and size to natural extracellular vesicles, and have active transmembrane protein structures, making them a good tool for delivering membrane protein biological signals. mEVs not only express most of the membrane proteins of the original cells, but also rarely destroy protein activity during the preparation process.

[0077] We selected the osteoblast cell line MLO-Y4 as seed cells and used adenovirus transfection to make it highly express Jagged-1 membrane protein. The specific process is as follows: Figure 1 and Figure 2 As shown in A. The transfection efficiency was evaluated by observing the fluorescence intensity 48 hours after adenovirus transfection. The expression of fluorescent protein indicated that the transfected target gene fragment had been successfully translated ( Figure 1 and Figure 2PCR results showed that adenovirus transfection successfully increased the expression level of Jagged-1 by nearly 3 times ( Figure 1 and Figure 2 (C) Meanwhile, the expression of Jagged-1 was also inhibited to 36.66% by transfection of interfering RNA.

[0078] Subsequently, after confirming that the transfection was effective, the cell membrane was extracted and engineered cell membrane vesicles were prepared by nanomembrane extrusion. Transmission electron microscopy (TEM) results showed that untransfected osteoblast vesicles (OMVs), Jagged1-overexpressing osteoblast vesicles (Jag1-OMVs) and Jagged1-expressing osteoblast vesicles (shJag1-OMVs) all exhibited a well-defined spherical cystic structure ( Figure 2 Nanoparticle tracking analysis showed that the peak nanosize of Jag1-OMVs was approximately 138.4 nm ( Figure 2 E). The membrane proteins of each group of vesicles were further extracted, and the expression of membrane proteins CD63, CD81, Jagged-1 and Na / K ATPase was analyzed by Western Blotting. The results showed that the expression of Jagged-1 in Jag1-OMVs was significantly increased, while CD63, CD81 and Na / K ATPase were expressed in the three groups of cell membrane vesicles ( Figure 2 Middle F).

[0079] To investigate whether Jag1-OMVs can activate the Notch pathway in osteocytes and improve vascular regulation, we used cell immunofluorescence to detect Notch pathway-related proteins. Figure 2 Middle G shows that the expression of Notch1 in osteocytes in the Jag1-OMVs group was significantly upregulated, and the expression of VEGFA also increased accordingly. Compared with the OMVs group, the expression of DLL1 (a downstream molecule of the Notch pathway) and osteogenic transcription factor Runx2 in the Jag1-OMVs group was also significantly enhanced, suggesting that osteocyte membrane vesicles can better complete the task of osteocyte activation rather than simply delivering Jagged1 ligands ( Figure 2 Middle H).

[0080] To further verify the positive activation of Jag1-OMVs on osteocytes, RT-PCR was used to analyze angiogenesis-related genes, including Notch1, NICD, VEGFA, HIF1α, and Ang2. The expression of these genes was significantly enhanced in the Jag1-OMVs group, while that of shJag1-OMVs was decreased ( Figure 2In addition, the gene expression trend of the osteogenic transcription factor Runx2 was consistent with the protein expression. Based on the above experimental results, we successfully prepared osteocyte membrane vesicles with stable structure and high expression of Jagged1 protein, which can promote the activation of Notch signaling and the expression of osteogenic transcription factors, which provides a basis for subsequent experiments.

[0081] 2. Preparation and characterization of mineralized hydrogel microspheres

[0082] The use of airflow control technology to prepare hydrogel microspheres loaded with lipid-soluble and highly bioactive drugs has unique advantages. First, compared with microfluidics, it has a fast flow rate, large output, and is more suitable for mass production; second, the airflow control preparation process is simple, which can avoid washing with organic reagents, thereby less damage to the activity of the load. Therefore, this study used the air shear microfluidics method to prepare hydrogel microspheres loaded with Jag1-OMVs to achieve the "freshness preservation" function of active proteins ( Figure 3 Gelatin (Gel) and sodium alginate (Alg) have high biosafety and have been widely used in the treatment of bone defects. Methacrylic anhydride gelatin (GelMA) and methacrylic anhydride alginate (AlgMA) were synthesized by modification. 1 The HNMR spectrum results showed that the characteristic peaks of MA were detected, indicating that the methacrylic acid groups were effectively incorporated ( Fig.10 ). Subsequently, the gas flow control technology precisely shears the microdroplets and passes through the Ca 2+ Ions cause the molecules of AlgMA and GelMA to fold and entangle to form hydrogel microspheres (which provide crystallization sites for the subsequent mineralization of hydrogel microspheres), and blue light cross-linking is performed to form a double network fiber structure. At the same time, sequential mineralization can introduce calcium phosphate crystals in situ into the three-dimensional fibers of the hydrogel, giving it a mineral composition similar to that of natural bone. We sequentially mineralized the hydrogel microspheres prepared by airflow control within 24 hours, such as Figure 3 As shown in B, terraced patterns formed on the surface of the microspheres after 0.5 h of mineralization, indicating that Ca 2+ The network formed by ions may gradually deconstruct. After 12 hours of mineralization, the transmittance of the microspheres under an optical microscope changed significantly, indicating mineral deposition. Mineralization was completed at 24 hours of mineralization, namely mineralized hydrogel microspheres (MHMs). MHMs were completely opaque, suggesting that a large amount of opaque mineral tissue was formed inside their fibers. To evaluate whether sequential mineralization affects the actual loading rate of the microspheres for Jag1-OMVs, we used laser scanning confocal microscopy (LSCM) to observe the fluorescence intensity. The results showed that Jag1-OMVs were well dispersed in the microspheres and had a high loading capacity ( Figure 3The diameter of MHMs is 134.21±23.6μm, which can be easily injected locally through a 21G needle ( Figure 3 D). The microstructure of MHMs was observed using a scanning electron microscope (SEM). The results showed that compared with HMs, MHMs had a multi-ridge structure with a large number of nanoscale crystal structures in the ridges ( Figure 3 E, G). Element scanning revealed that the calcium distribution of MHMs was nodular, which was different from the scattered Ca distribution of HMs, suggesting the formation of nano-calcium phosphate ( Figure 3 (F,H).

[0083] A good mineralization environment and matrix hardness are important for accelerating the transformation of bone cells. Moderate matrix hardness can promote osteogenic differentiation, while a softer matrix may be conducive to adipogenesis or other lineage differentiation. The optimal compression modulus of hydrogels that promote osteogenic lineage differentiation of stem cells is usually in the range of 5-40 kPa. This range simulates the mechanical properties of the natural bone microenvironment and is essential for guiding stem cells toward the osteogenic lineage. In order to determine the changes in the mechanical properties of mineralized microspheres, the dynamic mechanical method was used to measure the compression moduli of HMs, HMs mineralized for 12 hours (M12h), and MHMs, which were 0.04, 0.21, and 15.65 kPa, respectively. Figure 3 This suggests that MHMs can provide the ideal matrix stiffness for osteoblast transformation.

[0084] A good mineralization environment is very important for osteocyte differentiation. The main components of natural bone matrix are the coupling products of minerals and collagen. Mineral scaffolds are used in bone tissue engineering to achieve the generation of bionic implants, but large blocks of stable minerals are difficult to directly participate in the mineralization process of bone matrix, but require a series of internalization reconstruction. In order to explore the mineralization level and mineral composition of MHMs, we first used thermogravimetric analysis (TGA) to quantify the mineral content in mineralized hydrogel microspheres. Since calcium phosphate does not vaporize under high temperature conditions, heating the hydrogel microspheres to 800°C can remove the hydrogel without losing the mineral component. The results showed that the mineral percentages of HMs, M12h and MHMs were 8.38%, 27.94% and 45.85%, respectively, while the positive control ACP group was 91.89% ( Figure 3 J). FTIR spectrum at 560 and 940 cm -1 The phosphate band is shown at the MHMs, further indicating that calcium phosphate ( Figure 3 At the same time, the XRD analysis results showed that characteristic peaks appeared at angles of 26° and 32°, which suggested that the mineralized material in MHMs was mainly amorphous calcium phosphate ( Figure 3These characteristic peaks were absent in HMs and were most highly expressed in the ACP group. The above results indicate that our sequential mineralization method successfully formed mineralized hydrogel microspheres of amorphous calcium phosphate, which possessed matrix hardness suitable for osteogenic differentiation and an ideal mineralization environment.

[0085] 3. Biocompatibility and osteogenic ability of mineralized hydrogel microspheres

[0086] As a biomaterial for in vivo application, mineralized hydrogel microspheres need excellent biocompatibility. Therefore, the biocompatibility of mineralized hydrogel microspheres was tested by live-dead staining and CCK8. The experimental groups in this part were Control, HMs, MHMs, OMVs@MHMs and Jag1-OMVs@MHMs. Live-dead cell staining was performed after 3 days of co-culture ( Figure 4 In Figure 2A, BMSCs and HUVECs were tested respectively. The results showed that the cells had good morphology and viability, and there was no statistical difference between the groups. At the same time, the cell counting kit-8 (CCK-8) test on the third day showed that the proliferation rate was normal ( Figure 4 (middle B).

[0087] In addition, the osteogenic potential of mineralized hydrogel microspheres under co-culture conditions needs to be further identified. Under the induction of osteogenic conditioned medium, alkaline phosphatase (ALP) and alizarin red (ARS) staining were used to observe the osteogenic induction of each treatment group on the 5th and 14th days, respectively. The ALP staining results showed that compared with the Control group, the staining depth of the microsphere groups (HMs, MHMs, OMVs@MHMs and Jag1-OMVs@MHMs) was significantly improved ( Figure 5 This may be attributed to the presence of a certain concentration of Ca in the microspheres. 2+ ions. The results of Alizarin red staining showed that the mineralized hydrogel microspheres group had better calcium nodule deposition ability, and the Jag1-OMVs@MHMs group had the best effect ( Figure 5 (middle B).

[0088] The osteogenic capacity of the mineralized hydrogel microspheres was further evaluated using RT-PCR on day 7. Figure 5 In the middle C), the expression levels of early osteogenesis-related genes (Runx2, Osterix) were significantly upregulated in the Jag1-OMVs@MHMs group. At the same time, late osteogenesis-related genes such as OCN, OPN, and Col1 were significantly highly expressed in the mineralized hydrogel microsphere group. These results indicate that mineralized hydrogel microspheres can effectively provide a local microenvironment for osteogenic differentiation.

[0089] 4. Mineralized hydrogel microspheres promote osteoblast transformation and positive activation

[0090] The osteocyte network is a core role in controlling bone formation, bone resorption, hematopoietic balance and endocrine functions, allowing bones to adapt to environmental changes such as mechanical and hormonal changes in the body. Therefore, in the process of bone regeneration, early reconstruction of the osteocyte network is an effective way to achieve precise, orderly and intelligent bone regeneration. This study explored whether mineralized hydrogel microspheres can promote the transformation of osteocytes and positively activate vascular regulation. First, BMSCs cells were inoculated on mineralized hydrogel microspheres and cultured in osteogenic medium for 7 consecutive days. The expression changes of cell surface markers were observed by immunofluorescence ( Figure 4 The results showed that the osteoblast-loaded mineralized microspheres (OMVs@MHMs and Jag1-OMVs@MHMs) highly expressed the osteoblast markers DMP1 and Cx43, while the HMs group barely expressed them ( Figure 4 At the same time, PCR results also showed that osteoblast markers such as FGF23, E11, DMP1 and Cx43 were also upregulated at the transcriptional level ( Figure 4 Middle E).

[0091] Furthermore, in order to observe whether the cells on the microsphere surface were positively activated, we observed the expression levels of Notch1 and VEGFA by immunofluorescence. The results showed that compared with the Control group, the expression of Notch1 in Jag1-OMVs@MHMs increased by 5 times, and the expression of VEGFA increased by 4 times ( Fig.11 ).

[0092] Then, we placed the microspheres in the upper layer through the co-culture system and observed the tubule formation and migration ability of the endothelial cells in the lower layer. The tube formation experiment verified the angiogenesis ability of the mineralized hydrogel microspheres. Our results showed that there was basically no effective lumen development in the control group. After 6 hours, scattered punctate nodules or dendritic processes were mainly observed ( Figure 5 In the Jag1-OMVs@MHMs group, the Notch pathway of the cells on the surface of the microspheres was activated, which accelerated the formation of tubes and provided the possibility for early angiogenesis and osteogenesis. In addition, the degree of vascularization in the OMVs group was significantly higher than that in the control group ( Figure 5 F). The rapid migration of endothelial cells is also important. Therefore, we also performed a scratch test on the co-cultured cells to examine the effect of mineralized hydrogel microspheres on endothelial cell migration. The results showed that the Jag1-OMVs@MHMs group exhibited stronger migration activity due to the activation of the Notch pathway, which was higher than that of the other groups ( Figure 5 The subsequent PCR results also proved that the cells on the surface of the microspheres in the Jag1-OMVs@MHMs group highly expressed Notch1 and VEGFA mRNA ( Figure 5Therefore, the above results suggest that Jag1-OMVs@MHMs can effectively promote osteoblast transformation and positively activate its vascular regulation function.

[0093] 5. Mineralized hydrogel microspheres have no immune bone regeneration ability in vivo

[0094] The subcutaneous ectopic osteogenesis model has broad application prospects in tissue engineering. Unlike bone defect repair, it is less affected by factors such as inflammation and immunity. At the same time, since the subcutaneous tissue is not the native location of bone tissue, it lacks osteogenic stem cells and stimulating factors. Therefore, we used the subcutaneous ectopic osteogenesis model to evaluate the ability of mineralized hydrogel microspheres to promote osteocyte transformation and regulate bone regeneration. Figure 6 As shown in Figure A, we injected the mineralized hydrogel microspheres after in vitro osteogenic induction subcutaneously into the flanks of nude mice. The experimental groups were HMs, MHMs, OMVs@MHMs, and Jag1-OMVs@MHMs. To observe the early transformation of osteoblasts, samples were taken 4 weeks after injection. Interestingly, the surface of the tumor in the Jag1-OMVs@MHMs group showed quite extensive vascularization compared with the other groups ( Figure 6 (B). MicroCT analysis of the obtained bone blocks revealed that the BV, TV, and BV / TV of the Jag1-OMVs@MHMs group were increased to a certain extent. At the same time, the imaging images showed that the trabecular bone of the Jag1-OMVs@MHMs group had higher quality and more uniform distribution, and Tb.N, Tb.Th, and BMD were significantly increased ( Figure 6 (C). H&E histological analysis showed that there were a large number of mature osteocytes in the Jag1-OMVs@MHMs group, suggesting that it significantly promoted osteocyte differentiation. In contrast, the OMVs@MHMs group also had uniformly distributed dense cancellous bone matrix, but its cell morphology was vacuolar chondrocytes and hypertrophic chondrocytes ( Figure 6 (D). Masson staining analysis further confirmed the huge difference in bone maturity and mineralization status between the Jag1-OMVs@MHMs group and the control group. Immunofluorescence staining was used to observe the expression of osteogenic markers OPN and Runx2, and the results showed that the osteogenic promotion effect of the mineralized hydrogel sphere groups (MHMs, OMVs@MHMs, and Jag1-OMVs@MHMs) was very significant ( Figure 6 The vascular regulation markers VEGFA and CD31 were significantly upregulated in the Jag1-OMVs@MHMs group ( Figure 6 (F) In general, the subcutaneous ectopic osteogenesis experiment showed that Jag1-OMVs@MHMs accelerated early osteoblast differentiation, and the introduction of Jag1-OMVs significantly activated its vascular regulation ability, which provides hope for achieving ideal intelligent bone regeneration.

[0095] 6. In vivo therapeutic effect of mineralized hydrogel microspheres on large bone defects

[0096] To evaluate the efficacy of mineralized hydrogel microspheres in treating large bone defects in vivo, we created a rat model of severe (3 mm) bone defects at the femoral condyle and implanted composite hydrogel microspheres ( Figure 7 (A). The experimental groups were: Control, HMs, MHMs, OMVs@MHMs, and Jag1-OMVs@MHMs. The animals were sacrificed at 4 and 8 weeks after surgery, and the rats were reconstructed by Mciroct scanning. The results showed that at 4 weeks, there was almost no new bone development in the control group, while trabecular bone structure had appeared in the defect site of the Jag1-OMVs@MHMs group ( Figure 7 (B). Micro-CT reconstruction technology was used to reconstruct a 3D image of the defect site to more accurately observe the defect area. The results showed that the repair effect of the Jag1-OMVs@MHMs group was significantly better than that of the other groups at all time points ( Figure 7 Bone mineral density (BMD), bone volume / tissue volume (BV / TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N) were measured and analyzed in each group. Figure 7 As shown in D, the bone quality of the mineralized sphere group was significantly better than that of the control group and the ordinary hydrogel microspheres. At the same time, the quantitative data of trabecular bone in the Jag1-OMVs@MHMs group was better than that of other groups.

[0097] To further evaluate the osteogenic effect of mineralized hydrogel microspheres in vivo, bone tissue was stained with hematoxylin and eosin (HE) and Masson's trichrome ( Figure 8 (A). The results showed that in the blank control group, without any treatment, the defect was still connective tissue at 4 weeks, and it was not until 8 weeks that a large number of fibrous tissues filled with inflammatory cells appeared. At 8 weeks, more new bone grew in the treatment group, extending from the edge of the defect to the middle, almost filling the defect. It is worth noting that although the bone repair effect was good in the OMVs@MHMs group, the trabeculae at the defect site were scattered and there were still a large number of newly generated trabecular structures and fibrous tissues. In the Jag1-OMVs@MHMs group, the trabeculae were evenly distributed at 8 weeks, and no obvious fibrous tissue was observed, which suggests that the early transformation and activation of bone cells improves the efficiency of bone remodeling and makes the bone regeneration process more intelligent ( Figure 8 (in BE).

[0098] In addition, we performed IF analysis of OPN and OCN proteins at 4 and 8 weeks ( Fig. 9The results were consistent with the pathological staining. The expression levels of OCN and OPN in the Jag1-OMVs@MHMs group were higher than those in the Control, HMs, MHMs, and OMVs@MHMs groups, respectively. Subsequently, we observed the formation of h-type blood vessels by staining CD31 and Emcn. Immunofluorescence results also confirmed our expectations that the Jag1-OMVs@MHMs group could promote the high expression of CD31 in endothelial cells and promote the formation of h-type blood vessels ( Fig. 9 Interestingly, increased formation of h-type vessels was also observed in the OMVs@MHMs group, which may be due to the fact that the osteocyte membrane vesicles played a role in mimicking natural osteocytes ( Fig. 9 Middle C). Animal results of critical bone defects suggest that Jag1-OMVs@MHMs can promote intelligent bone regeneration by promoting osteocyte differentiation and activation and regulating angiogenesis.

[0099] Conclusion

[0100] The present invention innovatively prepares a mineralized hydrogel microsphere, which can provide the ideal mineralization environment and matrix hardness required for bone cell transformation to accelerate bone cell transformation. At the same time, the ability of bone cells to regulate the formation of vascular networks is positively activated by loading osteocyte membrane vesicles with high expression of JAG1. Ectopic bone regeneration experiments have shown that mineralized hydrogel microspheres can effectively promote the transformation and activation of bone cells in vivo. At the same time, in the rat femoral defect model, mineralized hydrogel microspheres also showed good regulation of osteogenesis and angiogenesis, greatly accelerating the bone healing process. This strategy provides a new perspective for the treatment of large clinical bone defects, while emphasizing the key role of bone cells, providing new thinking for the future treatment and basic research of orthopedic diseases.

[0101] Comparative Example 1

[0102] Referring to the method of Example 1, hydrogel microspheres were first prepared by gas shear microfluidic device. Aqueous phase solution: including 4wt% GelMA, 0.5wt% AlgMA, 0.1wt% Jag1-OMVs and 0.5wt% photosensitizer. Gas phase: nitrogen with a flow rate of 1L / min. The liquid phase caliber of the microfluidic device is 30G, and the gas phase caliber is 18G. Microdroplets were collected and cross-linked with a blue light for 15min. The collected hydrogel microspheres were then subjected to gradient mineralization, specifically in CaCl 2 and Na 3 PO 4 After being immersed in a sodium phosphate dihydrate solution (200 mmol / L) with shaking for 30 min, the microspheres were transferred to a calcium chloride solution and immersed in shaking for another 30 min. The gradient mineralization process of the hydrogel microspheres was completed by repeated immersion for 24 h.

[0103] The experimental results show that when the above method is used to prepare mineralized hydrogel microspheres, large hard matrices will be formed after the hydrogel is mineralized, which makes the microspheres non-injectable. On the other hand, the above method first prepares hydrogel microspheres and then performs gradient mineralization. This mineralization method forms mineralized substances in the pores of the hydrogel and cannot form amorphous calcium phosphate. However, the method of the present invention uses Ca in the first step. 2+ Ionic crosslinking allows the formation of calcium phosphate at the intersection of the hydrogel network rather than in the hydrogel pores, so the method of the present invention improves the firmness of the combination of calcium phosphate and hydrogel compared with the comparative example. In addition, the method of the present invention forms a large amount of amorphous calcium phosphate after in situ mineralization, while the comparative method cannot form amorphous calcium phosphate, and its effect on bone regeneration is limited. At the same time, the present invention uses a double-crosslinked hydrogel network to improve the stability of the hydrogel, so that it can maintain a stable structure in a phosphoric acid solution.

Claims

1. A method for preparing injectable mineralized hydrogel microspheres, characterized in that: The following steps are involved: (1) Cultivating bone cells in a basal medium to prepare a recombinant adenovirus, wherein the adenovirus vector contains coding regions for Jagged1, shJagged1, red fluorescent protein, and green fluorescent protein; then transfecting the bone cells with the adenovirus, collecting the total RNA after transfection, preparing cDNA by reverse transcription, collecting the bone cells, and preparing osteoblast membrane vesicles, which are recorded as Jag1-OMVs; (2) preparing methacrylic anhydride gelatin and methacrylic anhydride sodium alginate hydrogels, and then forming an aqueous phase solution with methacrylic anhydride gelatin, methacrylic anhydride sodium alginate, Jag1-OMVs and a photosensitizer, using nitrogen as the gas phase, and preparing hybrid hydrogel microspheres through a gas shear microfluidic device; (3) Using calcium chloride solution to collect microdroplets, and using blue light for cross-linking, the collected hydrogel microspheres are subjected to gradient mineralization to obtain the injectable mineralized hydrogel microspheres.

2. The preparation method according to claim 1, characterized in that: The bone cells in step (1) are cell line MLO-Y4, and the basal culture medium is a high-glucose DMEM culture medium containing 10 wt % fetal bovine serum.

3. The preparation method according to claim 1, characterized in that: In step (1), the bone cells were transfected with adenovirus for 24 hours, and the total RNA was collected 48 hours after transfection.

4. The preparation method according to claim 1, characterized in that: The preparation method of the methacrylic anhydride gelatin in step (2) is as follows: dissolve gelatin in water, add methacrylic acid dropwise at 4°C in the dark, mix evenly, then add NaOH solution, put the obtained liquid into a dialysis bag, dialyze and freeze-dry to obtain methacrylic anhydride gelatin; the preparation method of the methacrylic anhydride sodium alginate is as follows: dissolve sodium alginate in water, add methacrylic acid dropwise at 4°C in the dark, mix evenly, then add NaOH solution, put the obtained liquid into a dialysis bag, dialyze and freeze-dry to obtain methacrylic anhydride sodium alginate.

5. The preparation method according to claim 1, characterized in that: The weight ratio of methacrylic anhydride gelatin, methacrylic anhydride sodium alginate, Jag1-OMVs and photosensitizer in the aqueous solution in step (2) is 4:0.5:0.1:0.

5.

6. The preparation method according to claim 1, characterized in that: The flow rate of nitrogen in step (2) is 1 L / min.

7. The preparation method according to claim 1, characterized in that: In step (3), blue light cross-linking is performed for 15 minutes, and the concentration of the sodium phosphate dihydrate solution is 200 mmol / L.

8. The preparation method according to claim 1, characterized in that: The gradient mineralization process in step (3) is to oscillate and soak in a sodium phosphate dihydrate solution for 30 minutes, then transfer to a calcium chloride solution and continue to oscillate and soak for 30 minutes. The gradient mineralization process of the hydrogel microspheres is completed by repeated soaking for 24 hours.

9. Injectable mineralized hydrogel microspheres prepared by the method according to any one of claims 1 to 8.

10. Use of the injectable mineralized hydrogel microspheres according to claim 9 in the preparation of a drug for treating bone defect regeneration.

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