Hypoxia regulation exosome hydrogel as well as preparation method and application thereof in bone defect repair

Abstract

The invention provides hypoxia regulation exosome hydrogel and a preparation method and application thereof in bone defect repair, and compared with the prior art, the hypoxia regulation exosome hydrogel provided by the invention has the advantages that the exosome cultured by 3D stem cells modified by superparamagnetic ferroferric oxide nanoparticles is loaded on the GelMA hydrogel modified by bone targeting polypeptide, and the preparation method is simple. The intelligent oxygen-producing gel is innovatively constructed by skillfully utilizing the relationship between hypoxia and ROS generation according to the challenge that the hypoxia microenvironment of a bone defect part is closely followed. According to the hydrogel disclosed by the invention, osteogenesis regeneration is promoted by regulating and controlling a Wnt / beta-catenin pathway in osteoblasts, so that hypoxia and oxidative stress microenvironments are safely, efficiently and intelligently improved at injured parts. And the adhesion to the defect part is improved, and the problem that the superparamagnetic ferroferric oxide nanoparticles are difficult to effectively retain at the defect part is solved.

Description

Technical Field

[0001] The present invention relates to the fields of biomedical engineering and regenerative medicine, and in particular to a hypoxia-regulated exosome hydrogel and a preparation method thereof and an application thereof in bone defect repair. Background Art

[0002] Bone defects, especially critical size bone defects (CSBDs), are clinically difficult to treat. In addition, critical size bone defects refer to bone injuries that fail to heal spontaneously even after proper care and surgical stabilization. Currently, bone tissue engineering has the potential to provide a new therapeutic strategy that introduces an exogenous scaffold designed to promote cell growth and proliferation by providing regulatory growth factors. Studies have shown that bone damage and its subsequent chain reactions are significantly affected by the generation of free radicals, especially oxidative stress leading to the generation of reactive oxygen species (ROS), which impair the long-term stability of bones and implants and trigger osteoblast apoptosis. Previous studies have also demonstrated that oxidative stress is a major obstacle to bone repair. In contrast, ROS scavenging strategies have been shown to enhance osteoblast activity, thereby accelerating bone regeneration. However, an effective therapeutic strategy that simultaneously addresses oxidative stress to promote bone repair remains a challenge.

[0003] Bone transplantation and bone regeneration are both important therapeutic methods in the medical field, each with different characteristics and application scenarios. Traditional bone transplantation methods have problems such as insufficient donors and immune rejection. Current bone regeneration strategies are mainly divided into two categories: scaffolds implanted with exogenous stem cells, and scaffolds loaded with factors to recruit endogenous stem cells. Both methods promote bone regeneration through the paracrine effect of stem cells (SCs). It is worth noting that exosomes derived from stem cells (SC-Exo) play a key role in the paracrine process. Compared with stem cells and protein-based drugs, the inherent multicomponent properties of mesenchymal stem cell (MSCs)-derived exosomes (MSCs-Exo) trigger complex and persistent cellular responses. Many studies have confirmed that MSCs-Exo can enhance osteogenesis, angiogenesis, and bone mineralization, indicating that cell-free therapy based on MSCs-Exo has significant potential for bone defect repair. This approach provides a safe, economical, and efficient alternative that avoids immunological or ethical restrictions.

[0004] Exosomes are nanoscale vesicles secreted by cells, carrying a variety of bioactive molecules such as proteins, mRNA, miRNA, etc., and are involved in intercellular communication and material transport. Exosomes show regulatory effects in bone remodeling and fracture healing. It is suggested that exosomes can be used as a personalized therapeutic strategy to promote bone tissue repair. Although exosomes from bone marrow mesenchymal stem cells (BMSCs) have shown clear therapeutic potential, their efficacy is limited when cultured in traditional two-dimensional (2D) culture conditions of BMSCs. Compared with 2D monolayer culture, three-dimensional (3D) cultured MSC aggregates improve their biological properties, leading to improved anti-inflammatory, angiogenic and tissue regeneration effects. Therefore, 3D cultured MSCs-Exo (3D-Exo) has better therapeutic effects by transferring specific cargo. In particular, it has been demonstrated that 3D culture systems can increase the total yield of exosomes by more than 19 times compared with traditional 2D culture methods. Therefore, 3D cultured MSCs-Exo show higher yield and improved internalization activity, significantly enhancing the therapeutic effect and providing a new strategy for the repair of bone defects.

[0005] Tissue regeneration is a dynamic process involving interactions between cells and their surrounding matrix. This complex interaction can be significantly enhanced by biomaterial designs that can accommodate local cellular fluctuations, allowing them to modulate their properties in response to intrinsic biological signals. In addition, bone defects often occur simultaneously with disruption of local microvasculature, which presents a challenging scenario for bone regeneration, as bone regeneration is challenging in a hypoxic microenvironment caused by obstructed blood supply. However, research on sustained clearance of ROS during bone regeneration is still in its early stages and needs to be continued. Summary of the invention

[0006] The purpose of the present invention is to provide a hypoxia-regulated exosome hydrogel and a preparation method thereof. The present invention constructs a novel hypoxia-regulated exosome hydrogel, and the exosomes derived from 3D stem cell spheres modified by superparamagnetic ferroferric oxide nanoparticles are physically encapsulated in the GelMA hydrogel modified by the bone-targeted polypeptide. The activity of the superparamagnetic ferroferric oxide nanoparticles removes the ROS generated by the hypoxic microenvironment at the defect site, thereby relieving the hypoxic environment and promoting the bone repair ability of the exosomes derived from the 3D stem cell spheres. The osteogenic targeted polypeptide on the surface of the hydrogel is further used to increase the treatment time of the hypoxia-regulated exosomes and increase the adhesion of the hydrogel at the defect site. Therefore, the present invention obtains a new, efficient and intelligent bone defect repair material.

[0007] The specific technical solutions of the present invention are as follows:

[0008] A method for preparing a hypoxia-regulated exosome hydrogel comprises the following steps:

[0009] 1) Dissolve GelMA in PBS buffer, add maleimide polyethylene glycol active ester, heat and stir to react; then add HS-(DSS)6, heat and react to obtain Gel-DSS6 hydrogel solution;

[0010] 2) Azideacetic acid, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and superparamagnetic ferroferric oxide nanoparticle solution are mixed, and the mixture is stirred in an ice bath to react to obtain product 1; at the same time, dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester solution is added to the exosome solution 3D-Exo derived from three-dimensionally cultured bone marrow mesenchymal stem cell balls, and the mixture is stirred in an ice bath to react to obtain product 2; then, the reacted product 1 and product 2 are mixed, and the mixture is stirred in an ice bath; after the reaction is completed, ultrafiltration is performed to obtain hypoxia-regulated exosomes 3D-SExo;

[0011] 3) The 3D-SExo solution was added to a mixed solution of a photoinitiator solution and a Gel-DSS6 hydrogel solution, and then cured by ultraviolet light to prepare the hypoxia-regulated exosome hydrogel 3D-SExo / DGel.

[0012] In step 1), the dosage ratio of GelMA to PBS buffer is 0.15-0.25 g / mL;

[0013] In step 1), the mass ratio of GelMA, maleimide polyethylene glycol active ester and HS-(DSS)6 is (150-250):(0.8-1.2):(1.8-2.3);

[0014] In step 1), the step of adding maleimide polyethylene glycol active ester and heating and stirring the reaction means stirring the reaction in a water bath at 37° C. for 4 hours;

[0015] In step 1), the addition of HS-(DSS)6 and the heating reaction refers to stirring in a water bath at 37° C. for 4 h;

[0016] In step 2), the volume ratio of azidoacetic acid to superparamagnetic ferroferric oxide nanoparticle solution is 1:20; the concentration of superparamagnetic ferroferric oxide nanoparticle solution is 1.0 mg / mL, and the solvent is water;

[0017] In step 2), the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is 1:1;

[0018] In step 2), the dosage ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and azidoacetic acid is 0.02-0.03 mg / μL;

[0019] In step 2), the reaction was stirred in an ice bath for 4 h to obtain product 1;

[0020] In step 2), the volume ratio of the exosome solution to the dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester solution is (900-1100):(0.8-1.2); the solvent of the exosome solution is PBS buffer, wherein the concentration of the exosomes is 0.9-1.1 mg / mL; the solvent of the dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester solution is PBS buffer, wherein the concentration of the dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester is 4.9-5.1 mg / mL;

[0021] In step 2), the reaction was stirred in an ice bath for 4 h to obtain product 2;

[0022] In step 2), the volume ratio of the superparamagnetic ferroferric oxide nanoparticle solution to the exosome solution is: 0.8-1.2:0.9-1.1;

[0023] In step 2), product 1 and product 2 are mixed and stirred in an ice bath for 4 h;

[0024] In step 3), UV curing means: irradiating with a UV flashlight with a wavelength of 405 nm for 1 min;

[0025] In step 3), the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP).

[0026] In step 3), the volume ratio of the photoinitiator solution to the Gel-DSS6 hydrogel solution is 1:10;

[0027] In step 3), the volume ratio of 3D-SExo solution to Gel-DSS6 hydrogel solution is 1:1;

[0028] In step 3), the concentration of the photoinitiator solution is 2.5 mg / mL, and the solvent is PBS buffer solution; the 3D-SExo solution refers to 3D-SExo dissolved in PBS buffer solution with a concentration of 0.5 mg / mL; the Gel-DSS6 hydrogel solution refers to Gel-DSS6 dissolved in PBS buffer solution with a concentration of 0.2 g / mL.

[0029] The present invention provides a hypoxia regulating exosome hydrogel, which is prepared by the above method.

[0030] The present invention provides an application of a hypoxia-regulated exosome hydrogel in bone defect repair. The hypoxia-regulated exosome hydrogel is used to prepare a bone defect repair material. The hypoxia-regulated exosome hydrogel can be used to prepare a material for treating bone defects.

[0031] The design ideas of the present invention are as follows:

[0032] In the field of bone regeneration, superparamagnetic ferroferric oxide nanoparticles (SPION) can promote bone regeneration by enhancing osteogenesis and angiogenesis in bone models and have good reactive oxygen species (ROS) scavenging ability and biocompatibility. Based on the elevated ROS in the bone defect microenvironment, bone tissue engineering materials with ROS scavenging function are constructed, which can effectively promote bone regeneration and repair by promoting osteogenesis and / or inhibiting osteoclasts. Superparamagnetic ferroferric oxide nanoparticles are nanozymes with catalytic properties similar to peroxidase, catalase and superoxide dismutase, which can effectively decompose H2O2, thereby protecting cells from oxidative stress, and are also of great significance for human growth, development and metabolic activities. This property is suitable for ROS scavenging in the hypoxic microenvironment of bone defects. Therefore, the combination of 3D-Exo and SPION effectively promotes bone regeneration and scavenges ROS. In order to further improve the therapeutic efficacy of 3D-Exo, SPION is integrated into the surface of 3D-Exo through exosome engineering technology to obtain hypoxia-regulated exosomes, so as to achieve the purpose of promoting bone repair by alleviating the bone microenvironment. The engineered 3D-Exo exosomes deliver potent therapeutic molecules to bone defects and create an optimal microenvironment conducive to bone regeneration. Although most bone tissue engineering scaffolds have been shown to have clear osteogenic potential, supplementation with hypoxia-conditioned exosomes can enhance their performance.

[0033] GelMA is a photosensitive hydrogel with biocompatibility and biodegradability. The high water content of GelMA hydrogel provides a 3D environment similar to bone tissue and biocompatible for the culture of bone-related cells, and its internal relaxation environment is conducive to the formation of bone tissue. Bone-targeted peptide (DSS6) prefers to bind to low-crystalline hydroxyapatite and calcium phosphate on the surface of newly generated bone rather than existing bone tissue, increasing adhesion to the defect site. Therefore, DSS6-modified GelMA hydrogel scaffolds can prolong the retention time of hypoxia-regulated exosomes and further enhance their bone repair ability. Therefore, this hypoxia-regulated hydrogel provides new ideas and treatment methods for clinical bone defect repair.

[0034] The present invention provides a composition of 3D stem cell culture exosomes modified with superparamagnetic ferroferric oxide nanoparticles and GelMA hydrogel modified with bone-targeted peptides, which aims to solve the challenges in the treatment of critical size bone defects (CSBD). Through in vitro and in vivo experiments, the effectiveness of the hydrogel in promoting osteoblast differentiation, angiogenesis, and scavenging reactive oxygen species (ROS) has been demonstrated, providing a new, efficient, and intelligent treatment method for bone defect repair.

[0035] Compared with the prior art, the 3D stem cell-derived exosomes modified with superparamagnetic ferroferric oxide nanoparticles provided by the present invention are loaded on the GelMA hydrogel modified with bone-targeted polypeptide, and the preparation method is simple, low-cost and short-time. The present invention closely follows the great challenge of the hypoxic microenvironment at the bone defect site, and cleverly utilizes the relationship between hypoxia and ROS production to innovatively construct an intelligent oxygen-producing gel, which is expected to construct a new concept of bone tissue engineering materials. The hydrogel of the present invention promotes osteogenesis by regulating the Wnt / β-catenin pathway in osteoblasts, thereby safely, efficiently and intelligently improving the hypoxic and oxidative stress microenvironment at the site of injury. And the present invention increases the adhesion to the defect site, overcoming the problem that the engineered exosomes are difficult to stay in the defect site for a long time. The present invention provides an effective theoretical and practical basis, creating rich medical science and social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Representative SEM images of Gel-DSS6 hydrogel and 3D-SExo / DGel hydrogel;

[0037] Figure 2a The morphology of 3D-Exo and 3D-SExo observed by transmission electron microscopy (TEM);

[0038] Figure 2b is the size and potential of 3D-Exo and 3D-SExo;

[0039] Figure 3 is the Young's modulus of the Gel-DSS6 group and the 3D-SExo / DGel group;

[0040] Figure 4 To evaluate the decomposition ability of 3D-Exo, 3D-SExo and 3D-SExo / DGel hydrogels on H2O2;

[0041] Figure 5 is the cumulative release of 3D-Exo from different hydrogels;

[0042] Figure 6a Representative images of HUVECs migration and tube formation experiments under different treatments;

[0043] Figure 6b are the number of migrated cells (n=3), the number of tubes with different treatments (n=3), the total number of connections (n=3), and the total length (n=3);

[0044] Figure 7a Heat map of differentially expressed mirna between 2D-Exo and 3D-Exo;

[0045] Figure 7bThe enrichment of differentially expressed miRNAs between 2D-Exo and 3D-Exo in the KEGG pathway. The size of the dots shows the number of differentially expressed genes in this pathway. The larger the enrichment factor value, the greater the KEGG enrichment degree.

[0046] Figure 7c Representative images of ALP staining of BMSCs differentiated into osteoblasts after different treatments and representative images of Alizarin red staining of BMSCs differentiated into osteoblasts after different treatments;

[0047] Figure 7d For quantitative analysis of positive expression;

[0048] Figure 8 Representative images of immunofluorescence staining of OPN, NRF2, Runx2, α-SMA, CD31 and β-catenin in the skull defect area of ​​mice after 8 weeks of different treatments (yellow arrows point to the periosteum). DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0051] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0052] The reagents and instruments used in the present invention are as follows:

[0053] The PBS buffer used in the present invention is 0.1M, pH=7.4 PBS buffer;

[0054] Methacryloyl gelatin (GelMA) (Engineering for life, Jiangsu, China);

[0055] Maleimide polyethylene glycol active ester;

[0056] bone-targeting peptide HS-(DSS)6 (Taopu Biotechnology, Jiangsu, China);

[0057] Azidoacetic acid;

[0058] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0059] N-hydroxysuccinimide;

[0060] Superparamagnetic Fe3O4 nanoparticles (Donna Biotechnology, Nanjing, China);

[0061] Dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester;

[0062] cell membrane deep red fluorescent dye (KGMP0026, KeyGen Biotech, Jiangsu, China);

[0063] transmission electron microscope (HT-7800, Hitachi, Tokyo, Japan);

[0064] nanoparticle tracking analyzer (Particle Metrix, Munich, Germany);

[0065] Scanning electron microscope (sigma300, Zeiss, Germany), energy dispersive spectrometer: (Xplore30 Oxford Instruments, Oxford, UK);

[0066] Universal Mechanical Testing System (68SC, Instrom, Boston, USA);

[0067] Microplate reader (Flexstation 3, California, USA);

[0068] In vivo imaging system (IVIS LuminaLT * , PerkinElmer, Massachusetts, USA);

[0069] microcomputed tomography (Skyscan 1276, Brooke, MA, USA);

[0070] Inverted microscope (Nikon, Tokyo, Japan).

[0071] Example 1

[0072] Preparation of 2D-Exo from Mesenchymal Stem Cells:

[0073] Will Mouse bone marrow mesenchymal stem cells were cultured in a petri dish using The cells were cultured in complete medium of mouse bone marrow mesenchymal stem cells. When the cell density grew to 80%, the complete medium was replaced with exosome-free medium, and the supernatant was collected to separate 2D-Exo.

[0074] Preparation of 3D cultured mesenchymal stem cell exosomes (3D-Exo), preparation of 3D bone marrow mesenchymal stem cell aggregates using the droplet overlay method:

[0075] 70 μl of 5 mg / mL agarose matrix prepared with 0.1 M, pH 7.4 PBS buffer was spread on the bottom of a 96-well plate. Mouse bone marrow mesenchymal stem cells were 10 4 Each well was seeded with agarose matrix in a 96-well plate. Complete medium of mouse bone marrow mesenchymal stem cells was maintained for one week, and then bone marrow mesenchymal stem cell aggregates were obtained. To collect exosomes derived from bone marrow mesenchymal stem cell aggregates, the complete medium was replaced with exosome-free medium, and the supernatant was collected to isolate 3D-Exo.

[0076] Isolation of Exo using ultracentrifugation:

[0077] The supernatant of bone marrow mesenchymal stem cells and aggregates was collected, and cells and cell debris were first removed by centrifugation at 10,000 × g at 4°C for 20 minutes. Then, larger extracellular vesicles were discarded by centrifugation at 100,000 × g at 4°C for 90 minutes. Finally, precipitated 2D-Exo and 3D-Exo were obtained.

[0078] A method for preparing a hypoxia-regulated exosome hydrogel comprises the following steps:

[0079] 1) Dissolve 0.2 g of GelMA in 1 mL of 0.1 M, pH = 7.4 PBS buffer, then add 1 mg of maleimide polyethylene glycol active ester, and stir in a water bath at 37° C. for 4 hours; then, add 2 mg of bone targeting polypeptide and stir in a water bath at 37° C. for 4 hours to obtain a Gel-DSS6 hydrogel solution;

[0080] 2) 50 μl of azidoacetic acid, 1.5 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1.5 mg of N-hydroxysuccinimide and 1 mL of 1.0 mg / mL superparamagnetic ferroferric oxide nanoparticle aqueous solution was mixed and stirred in an ice bath for 4 hours as product 1; at the same time, the exosomes purified by ultracentrifugation were prepared into 1 mg / mL exosome solution 1 mL with 0.1 M, pH = 7.4 PBS buffer, and 1 μl of 5 mg / mL dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester solution prepared with 0.1 M, pH = 7.4 PBS buffer was added, and stirred in an ice bath for 4 hours as product 2; then, the reacted products 1 and 2 were mixed and stirred in an ice bath for 4 hours; after the reaction was completed, ultrafiltration was performed using a 100KD ultrafiltration centrifuge tube at 4°C, 5000g, and 10min. After completion, PBS was added to continue ultrafiltration, and the reaction was repeated 3 times to remove the residual azidoacetic acid to obtain 3D-SExo;

[0081] 3) 3D-SExo was dissolved in PBS buffer to obtain a 3D-SExo solution with a concentration of 0.5 mg / mL; Gel-DSS6 was dissolved in PBS buffer to obtain a Gel-DSS6 hydrogel solution with a concentration of 0.2 g / mL; the photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate was dissolved in PBS buffer solution to obtain an initiator solution with a concentration of 2.5 mg / mL; 30 μL of 0.2 g / mL Gel-DSS6 hydrogel solution was mixed with 3 μL of 2.5 mg / mL initiator solution, 30 μL of 0.5 mg / mL 3D-SExo solution was added to the obtained mixed solution, and an ultraviolet flashlight with a wavelength of 405 nm was irradiated for 1 min to prepare hypoxia-regulating exosome hydrogel 3D-SExo / DGel (3D-SExo / DGel composite hydrogel).

[0082] Electron microscopy characterization of Gel-DSS6 and 3D-SExo / DGel:

[0083] like Figure 1 As shown, scanning electron microscopy (SEM) revealed that the surface of Gel-DSS6 was uneven and wavy, and round exosomes were attached to 3D-SExo / DGel.

[0084] Characterization of transmission electron microscopy analysis (3D-Exo and 3D-SExo):

[0085] The morphology of nanoparticles 3D-Exo and 3D-SExo was observed by transmission electron microscopy at 100 kV accelerating voltage. The concentration and size of exosomes were measured using a nanoparticle tracking analyzer. Gel-DSS6 hydrogel and 3D-SExo / DGel composite hydrogel were dried using a freeze dryer. The morphology of the sample surface was observed by scanning electron microscopy using platinum spray at 20 mA for 45 s.

[0086] The appearance of 3D-Exo and 3D-SExo is as follows Figure 2a As shown, transmission electron microscopy (TEM) analysis showed that the morphology of 3D-Exo and 3D-SExo was spherical and well-dispersed in appearance. Figure 2b The nanoparticles shown showed a uniform size distribution, with the 3D-Exo particle size being approximately 80 nm and the 3D-SExo average particle size being approximately 100 nm. Zeta potential measurements showed that the negative charge on the surface of the nanoparticles was beneficial for improving biocompatibility.

[0087] Mechanical testing to investigate the mechanical properties of composite hydrogels:

[0088] To test the mechanical properties of the composite hydrogels, long strip samples (length, 15 mm; width, 5 mm; height, 1 mm) of Gel-DSS6 and 3D-SExo / DGel hydrogels were prepared and then subjected to compression tests, which were calculated from the peak values ​​in the stress-strain curves obtained by a universal mechanical testing system.

[0089] Through mechanical testing, mechanical test results Figure 3 It shows that the Young's modulus of the composite hydrogel is relatively reduced after the addition of 3D-SExo (but the strength does not decrease significantly), indicating that the composite hydrogel has good flexibility.

[0090] Evaluation of ROS scavenging by 3D-SExo / DGel hydrogels:

[0091] Using PBS buffer solution as solvent, 30 μL of 0.5 mg / mL 3D-Exo solution and 30 μL of 0.5 mg / mL 3D-SExo solution were prepared; and the 3D-SExo / DGel composite hydrogel prepared in Example 1 was also prepared. 30 μL of 0.88 M H2O2 aqueous solution was added to each group and incubated for 12 hours. After adding ROS probe for co-incubation, the fluorescence intensity of the solutions in different groups was detected using an ELISA instrument. The scavenging ability of 3D-SExo / DGel hydrogel on H2O2 was evaluated.

[0092] like Figure 4 As shown, the H2O2 clearance rate of 3D-SExo / DGel was the highest compared with that of 3D-Exo and 3D-SExo, indicating that it had the highest ROS clearance efficiency in a short time.

[0093] In vitro release characteristics determination:

[0094] In vitro release studies were conducted in PBS buffer (0.1M, pH=7.4) solution, and DiR was used to label exosomes to monitor their release from 3D-SExo / DGel in real time. The specific experimental process was: 60μl PBS was added to 30μL 0.5mg / mL 3D-Exo solution, 30μL 0.5mg / mL 3D-SExo solution, and 3D-SExo / DGel composite hydrogel prepared according to Example 1, and incubated in a 37°C constant temperature incubator. The supernatant at the specified time point was collected, and the release rate of exosomes was detected by IVIS.

[0095] like Figure 5 As shown, compared with 3D-Exo and 3D-SExo, the 3D-Exo release time of 3D-SExo / DGel was significantly prolonged, indicating that Gel-DSS6 can effectively prolong the release of 3D-Exo.

[0096] Detection of the angiogenesis ability of HUVECs in vitro by 3D-SExo / DGel:

[0097] In order to detect the effect of H2O2 on HUVECs migration in the hypoxic microenvironment in the anaerobic system, the Transwell assay was used. 4 HUVECs cells were implanted into the upper chamber of the Transwell. Then 50 μL of 0.5 mg / mL 3D-Exo solution and 50 μL of 0.5 mg / mL 3D-SExo solution were added. In addition, 50 μL of Gel-DSS6 hydrogel was mixed with 5 μL of 2.5 mg / mL initiator solution, and 50 μL of 0.2 g / mL Gel-DSS6 hydrogel solution was mixed with 5 μL of 2.5 mg / mL initiator solution according to the method of Example 1. 50 μL of 0.5 mg / mL 3D-SExo solution was added to the resulting mixed solution, and irradiated with a UV flashlight with a wavelength of 405 nm for 1 min to prepare 3D-SExo / DGel composite hydrogels, which were added to the lower layer of the 24-well plate respectively; in addition, no substance was added as the control group Con group. The plate was then treated to an anaerobic state. After 6 hours, the chamber was fixed with 4% paraformaldehyde and then stained with crystal violet solution. Gently wipe the cells on the upper surface of the membrane with a cotton swab and observe the cells on the lower surface of the membrane under a microscope.

[0098] 500 μL of HUVECs were added at 5 × 10 4 The concentrations of Matrix-Gel were inoculated in 24-well plates, and the tube formation was detected in each group, including Con group, Gel-DSS6 group, 3D-Exo group, 3D-SExo group, and 3D-SExo / DGel group. TM Matrigel. The plate was then incubated in an anaerobic system to form tubes. After 6 h of culture, HUVECs were photographed under a microscope and the number of tubes was counted.

[0099] First, dye Figure 6aThe results showed that 3D-SExo / DGel could promote the migration of HUVECs under hypoxia and induce HUVECs to form a primary vascular-like network structure composed of tubular structures. In the results of 6b, the number of migrating cells in the 3D-SExo group was about 2 times higher than that in the Con group and the 3D-Exo group, but still lower than that in the 3D-SExo / DGel group. At the same time, it can be seen that in the 3D-SExo group and the 3D-SExo / DGel group, a large number of primary vascular-like network structures composed of tubular structures radiating outward from cell aggregates were induced, and the number of tubular structures in the 3D-SExo / DGel group was greater than that in the 3D-SExo group. Only incomplete tube formation was seen in the Con group and the Gel-DSS6 group. It can be seen that the 3D-SExo / DGel hydrogel group has the ability to promote tube formation, and at the same time shows higher cell migration rate and vascular density in the bone defect area.

[0100] In vitro osteoblast differentiation and mineralization capacity:

[0101] The difference of miRNA between 2D-Exo and 3D-Exo was detected by miRNA sequencing (such as Figure 7a ), removed miRNAs with relatively low abundance, and selected 5 up-regulated miRNAs and 10 down-regulated miRNAs. Among the up-regulated miRNAs, miR-122-5p was shown to promote osteogenesis. In addition, the KEGG pathway enrichment map showed that the up-regulated miRNAs were enriched in the Wnt and TGF-β pathways, which regulate cell differentiation and proliferation, respectively (e.g. Figure 7b ).

[0102] BMSC cells were seeded in six-well plates, and RNATransMate / siRNA complexes were prepared according to the supplier's instructions. MiR-122-5p was overexpressed and divided into Con group, 3D-SExo / DGel group, miR-122-5P group, miR-122-5P+SPION group, and miR-122-5P+3D-SExo / DGel group. Among them, miR-122-5p was added to the miR-122-5P group, miR-122-5P+SPION group, and miR-122-5P+3D-SExo / DGel group. Subsequently, 50 μl of 0.25 mg / ml SPION prepared with PBS buffer was added to the miR-122-5P+SPION group, and D-SExo / DGel composite hydrogel prepared according to the method of Example 1 was added to the 3D-SExo / DGel group and the miR-122-5P+3D-SExo / DGel group (50 μL of 0.2 g / mL Gel-DSS6 hydrogel solution was mixed with 5 μL of 2.5 mg / mL initiator solution, and 50 μL of 0.5 mg / mL 3D-SExo solution was added to the resulting mixed solution, and irradiated with a UV flashlight with a wavelength of 405 nm for 1 min to prepare 3D-SExo / DGel composite hydrogel), and continuously co-cultured with various materials in an anaerobic system for osteogenic induction. When the cell confluence reached 70%, the cells were used Mouse BMSC Osteogenic Differentiation Kit. Change differentiation medium every 2 days.

[0103] ALP staining: After 3 days of co-culture induction, the cells were washed with PBS, fixed with 4% PFA for 30 minutes, and ALP activity was detected using an ALP staining kit. BMSCs were incubated with ALP colorimetric reagent for 30 minutes in the dark, and the results were observed under a microscope. Alizarin red staining: After 7 days of co-culture, the cells were washed with PBS, fixed with 4% PFA for 30 minutes, and ARS stained for 15 minutes, and calcium nodules were observed under a camera and microscope.

[0104] like Figure 7c As shown in the figure, alkaline phosphatase (ALP) and mineralized nodule (ARS) staining confirmed that the osteoblast differentiation and mineralization ability of the hydrogel group were significantly enhanced. In order to further verify the effect of 3D-SExo / DGel on the entire osteogenesis cycle, ALP staining and Alizarin red staining were used to detect early osteogenic differentiation and late calcium nodule formation, respectively.

[0105] like Figure 7dAs shown, after 3 days, it can be found that the number of ALP-positive BMSCs in the miR-122-5P group or miR-122-5P+SPION group was 2 times higher than that in the Con group and slightly lower than that in the 3D-SExo / DGel group without transfection of miR-122-5p. In addition, the ALP staining results of the 3D-SExo / DGel group were better than those of the Con group. At the same time, the ALP positivity rate of BMSCs in the miR-122-5P+3D-SExo / DGel group was further increased, indicating that miR-122-5p can increase the expression of ALP in early osteogenic differentiation. In addition, a similar trend can be found in the late stage of osteogenic induction by Alizarin red staining. Through these experimental results, it was found that the continuous and stable removal of reactive oxygen species in a hypoxic environment can promote the osteogenic differentiation of osteoblasts, confirming that the osteoblast differentiation and mineralization ability of 3D-SExo / DGel hydrogels were significantly enhanced, and miR-122-5p could also significantly enhance the formation of mineralized matrix in the Con group and 3D-SExo / DGel group.

[0106] In summary, the present invention develops SPION-modified 3D cultured MSCs-Exo, called 3D-SExo / DGel, for promoting bone regeneration, ROS scavenging and vascularization to meet the needs of bone defect areas. After encapsulation in GelMA modified with bone-targeting peptides, a composite hydrogel (3D-SExo / DGel) was obtained, which can prolong the retention time of exosomes. In addition, the difference in miRNA between 2D-Exo and 3D-Exo was detected by miRNA sequencing to better understand the mechanism and key signaling pathways of osteogenic differentiation induced by 3D-SExo / DGel, and further verified by miRNA overexpression transfection. After 3D-SExo / DGel was implanted into the bone defect area, 3D-SExo / DGel decomposed ROS to reduce the inhibitory effect of ROS on β-catenin production, and 3D-SExo / DGel released miRNA (miR-122-5p) to activate the Wnt / β-catenin pathway, thereby increasing the content of β-catenin and effectively promoting bone regeneration.

[0107] The present invention also conducted an immunofluorescence analysis study on 3D-SExo / DGel promoting the repair of mouse skull defects:

[0108] Mice were housed individually in separate cages for one week to acclimate to the environment. Anesthesia was induced by inhalation of ether. A circular full-thickness bone defect with a diameter of 2.5 mm was created on the top of the head. Subsequently, 30 μL of 0.5 mg / mL 3D-Exo solution and 30 μL of 0.5 mg / mL 3D-SExo solution were implanted into the defect area of ​​mice, respectively; in addition, 30 μL of Gel-DSS6 hydrogel was mixed with 3 μL of 2.5 mg / mL initiator solution, added to the defect area of ​​mice, and irradiated with a UV flashlight with a wavelength of 405 nm for 1 min to form gel in situ; and according to the method of Example 1, 30 μL of 0.2 g / mL Gel-DSS6 hydrogel solution was mixed with 3 μL of 2.5 mg / mL initiator solution, 30 μL of 0.5 mg / mL 3D-SExo solution was added to the obtained mixed solution, added to the defect area of ​​mice, and irradiated with a UV flashlight with a wavelength of 405 nm for 1 min to form gel in situ; in addition, the control group Con group without adding any substance was used to obtain mouse skull specimens at 4 weeks and 8 weeks after surgery. The specimens were fixed in 4% paraformaldehyde, and micro-computed tomography was then used to assess the regeneration of the defect area. The calvarial defect area after 3D-SExo / DGel implantation was sectioned and immunofluorescence was performed using antibodies (OPN, NRF2, Runx2, α-SMA, CD31, β-catenin).

[0109] like Figure 8 Through OPN immunofluorescence staining, a large amount of rope-like green fluorescence was observed in the 3D-SExo / DGel group, forming a lamellar bone-like structure. Correspondingly, the band-like green fluorescence in the 3D-SExo group and the 3D-Exo group was significantly reduced, and there was almost no similar fluorescence in the Con group. Through NRF2 immunofluorescence staining, it was known that the NRF2 immunofluorescence expression (green) in the Con group was significantly higher than that in other groups, indicating that the cells were in an anti-oxidative stress state; the Runx2 immunofluorescence expression (red) in the 3D-SExo / DGel group was significantly higher than that in other groups, indicating that its bone formation ability was strong; the CD31 immunofluorescence expression (red) in the 3D-SExo group was significantly higher than that in other groups, indicating that its tube formation ability was good.

[0110] In summary, the hypoxia-regulated exosome hydrogel provided by the present invention comprises exosomes cultured in 3D stem cells modified with superparamagnetic ferroferric oxide nanoparticles, and GelMA hydrogel modified with bone-targeted polypeptides. It increases adhesion to the defect site by encapsulating exosomes and superparamagnetic ferroferric oxide nanoparticles, and utilizing the osteogenic targeting polypeptide on the surface of the hydrogel. At the same time, superparamagnetic ferroferric oxide nanoparticles have the ability to remove ROS generated by the hypoxic microenvironment at the defect site. It can promote osteogenesis by regulating the Wnt / β-catenin pathway in osteoblasts. The hypoxia-regulated exosome hydrogel is used to prepare materials including but not limited to vertebral compression fractures, skull defects, jaw defects, long bone defects, pelvic defects, bone defects in spinal fusion, bone defects after bone tumor resection, congenital bone deformities, bone defect repair and reconstruction, and any other materials requiring bone tissue repair.

[0111] The technical solution of the present invention is the inventor's scientific research project, project name: Hypoxia-regulated exosome hydrogel promotes bone defect repair and mechanism research, project number 2023jc22.

[0112] The description of the above 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 protection scope of the present invention.

Claims

1. A method for preparing a hypoxia-regulated exosome hydrogel, characterized in that: The preparation method comprises the following steps: 1) Dissolve GelMA in PBS buffer, add maleimide polyethylene glycol active ester, heat and stir to react; then add HS-(DSS)6, heat and react to obtain Gel-DSS6 hydrogel solution; 2) Azideacetic acid, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and superparamagnetic ferroferric oxide nanoparticle solution are mixed, and the mixture is stirred in an ice bath to react to obtain product 1; at the same time, dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester solution is added to the exosome solution 3D-Exo derived from three-dimensionally cultured bone marrow mesenchymal stem cell balls, and the mixture is stirred in an ice bath to react to obtain product 2; then, the reacted product 1 and product 2 are mixed, and the mixture is stirred in an ice bath; after the reaction is completed, ultrafiltration is performed to obtain hypoxia-regulated exosomes 3D-SExo; 3) The 3D-SExo solution was added to a mixed solution of a photoinitiator solution and a Gel-DSS6 hydrogel solution, and then cured by ultraviolet light to prepare the hypoxia-regulated exosome hydrogel 3D-SExo / DGel.

2. The preparation method according to claim 1, characterized in that: In step 1), the mass ratio of the GelMA hydrogel, maleimide polyethylene glycol active ester and HS-(DSS)6 is (150-250): (0.8-1.2):(1.8-2.3)。 3. The preparation method according to claim 1 or 2, characterized in that: In step 1), the adding of maleimide polyethylene glycol active ester and heating and stirring the reaction refers to stirring the reaction in a water bath at 37° C. for 4 hours.

4. The preparation method according to claim 1, characterized in that: In step 1), the addition of HS-(DSS)6 and the heating reaction refer to stirring in a water bath at 37° C. for 4 hours.

5. The preparation method according to claim 1, characterized in that: In step 2), the volume ratio of azidoacetic acid to superparamagnetic ferroferric oxide nanoparticle solution is 1:20; the concentration of superparamagnetic ferroferric oxide nanoparticle solution is 1.0 mg / mL; the mass ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride to N-hydroxysuccinimide is 1:1; the amount ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride to azidoacetic acid is 0.02-0.03 mg / μL.

6. The preparation method according to claim 1 or 5, characterized in that: In step 2), the volume ratio of the superparamagnetic ferroferric oxide nanoparticle solution to the exosome solution is 0.8-1.2:0.9-1.

1.

7. The preparation method according to claim 1 or 6, characterized in that: In step 2), the volume concentration of the exosome solution and the dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester solution is (900-1100):(0.8-1.2).

8. The preparation method according to claim 1 or 6, characterized in that: In step 2), the reaction was stirred in an ice bath for 4 hours to obtain product 1; the reaction was stirred in an ice bath for 4 hours to obtain product 2; product 1 and product 2 were mixed and reacted in an ice bath for 4 hours.

9. A hypoxia-regulated exosome hydrogel prepared by the method according to any one of claims 1 to 8.

10. Use of the hypoxia-regulated exosome hydrogel according to claim 9 in bone defect repair, characterized in that: The hypoxia-regulated exosome hydrogel is used to prepare bone defect repair materials.

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