Antler-dcb@evsabpcs bone graft material and preparation method

Abstract

The application discloses a preparation method of a deer antler-DCB@EVsABPCs bone graft material, and specifically comprises the following steps: step 1, preparing an Antler-DCB support; step 2, preparing a 10% HAMA hydrogel and ABPCs-EVs; step 3, mixing; step 4, taking the Antler-DCB and immersing it into the HAMA hydrogel containing the ABPCs-EVs; and step 5, exposing to 405 nm ultraviolet light for 30 seconds to obtain the bone graft material. The application further discloses the deer antler-DCB@EVsABPCs bone graft material. The deer antler-DCB@EVsABPCs bone graft material and the preparation method thereof disclosed by the application have a strong ability to regulate osteogenesis, angiogenesis, neurogenesis and immune response, thereby providing a potential therapeutic intervention measure for treating serious bone defects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical material preparation technology, specifically relating to a method for preparing antler-DCB@EVsABPCs bone graft material, and also relating to antler-DCB@EVsABPCs bone graft material. Background Technology

[0002] Bone injury is one of the most common disabling injuries worldwide, affecting over 400 million patients with acute or long-term consequences that severely impact their quality of life. Bone regeneration is not a single, isolated process of bone growth, but a complex and dynamic one involving the coordinated growth of skeletal, vascular, and neural structures, complicated by a powerful immune response. Significant progress has been made in the development of various bone grafts over the past decade through the integration of cells, growth factors, and biomaterials. However, the clinical integration rate of engineered bone grafts remains low, and the lack of a single-use bone graft construction strategy leaves unresolved challenges in achieving effective bone regeneration. Existing biomedical materials fail to meet the practical requirements of low immunogenicity, high safety, high repair rates, and high bioactivity. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing antler-DCB@EVsABPCs bone graft material, which solves the problems of immune rejection and insufficient bioactivity in existing natural bone grafts.

[0004] Another object of the present invention is to provide antler-DCB@ABPCs-EVs bone graft material.

[0005] The first technical solution adopted in this invention is a method for preparing antler-DCB@EVsABPCs bone graft material, which is implemented according to the following steps:

[0006] Step 1: Fabrication of the Antler-DCB scaffold;

[0007] Step 2: Prepare a 10% HAMA hydrogel with ABPCs-Evs;

[0008] Step 3: Take an equal volume of hydrogel containing 10% HAMA and mix it with ABPCs-EVs;

[0009] Step 4: Immerse Antler-DCB in a 10% HAMA hydrogel containing ABPCs-EVs for 30 minutes;

[0010] Step 5: Remove the Antler-DCB containing 10% HAMA hydrogel and expose it to 405nm ultraviolet light for 30 seconds to obtain DCB@ABPCs-EVs bone graft material, and freeze it at -80℃.

[0011] The first technical solution of the present invention is further characterized in that step 1 specifically comprises the following steps:

[0012] S1, cut the cancellous bone of the antler into 4×4mm bone samples, repeatedly freeze and thaw, and then treat with ultrasound;

[0013] S2, the bone sample after S1 ultrasonic treatment is immersed in ethanol and shaken on a shaker for 3 hours;

[0014] S3, Immerse the bone sample soaked in S2 in ether and shake on a shaker for 1.5 hours;

[0015] S4. The bone sample soaked in S3 was immersed in a sodium hydroxide solution with a mass concentration of 20 g / L and shaken on a shaker for 12 hours. After soaking, the bone sample was washed with running water for 3 hours. After washing, it was treated with hydrogen peroxide with a mass fraction of 30% for 24 hours. After treatment, it was washed again.

[0016] S5. After cleaning, the bone sample is placed in a mixture of chloroform and methanol at a volume ratio of 3:1 for 1 hour. The bone sample is then rinsed with distilled water and dried at 70°C for 1 day to produce Antler-DCB.

[0017] The ultrasonic treatment in S1 specifically involves treating the bone sample with 40kHz ultrasound for 20 minutes.

[0018] The specific steps for the second cleaning in S4 are as follows: wash the bone sample three times with running water for 0.5 hours each time, then soak the bone sample in a DNase solution containing 1 mg / mL for 48 hours, and after the second cleaning, soak the sample in a solution containing 0.5 mg / mL RNase for 48 hours, and wash it three times with running water for 0.5 hours each time.

[0019] The specific steps for preparing the HAMA hydrogel in step 2 are as follows:

[0020] A1. Take 10 mL of PBS buffer and add it to a bottle containing 0.025 g of LAP. Heat in a water bath at 40-50 °C for 15 minutes, shaking several times during the process, to prepare the initiator standard solution.

[0021] A2. Take the required mass of methacrylamide hyaluronic acid and put it into a container; take the required volume of initiator standard solution and add it to the container containing methacrylamide hyaluronic acid. Stir and dissolve at room temperature for 0.5-1 h, then heat the solution to 80°C for 30 minutes; then transfer it to an ice-water mixture and soak for 5 min to obtain HAMA hydrogel.

[0022] The HAMA hydrogel in A2 consists of HAMA, lithium phenyl phosphate, and deionized water, with a volume ratio of 10:0.25:89.75.

[0023] The specific steps for preparing ABPCs-Evs in step 2 are as follows:

[0024] B1, obtain embryonic tissue 3-7 days after the antlers fall off;

[0025] B2, cut the germ tissue into 0.2 mm square pieces;

[0026] B3. Wash the cut tissue blocks three times with PBS buffer.

[0027] B4. Place the cleaned tissue blocks into a digestion solution with a volume ratio of type I: type II: type III collagenase of 1:1:1 and digest at 37°C for 30 minutes.

[0028] B5. The digested tissue block was transferred to a 10 cm culture dish for primary cell culture. When the cell density reached 80%, flow cytometry was performed to separate the cells.

[0029] B6. Cells separated from B5 were co-incubated with antibodies CD31, CD45, and CD235a, and then flow cytometry was performed to collect cells containing CD31-CD45-CD235a-.

[0030] B7. Collect cells containing CD31-CD45-CD235a- from B6, digest and centrifuge them, incubate the separated cells with ABPC marker antibodies CX43 and FGFR2, and perform flow cytometry sorting to collect ABPCs. Then, culture them in DMEM complete medium containing 10% FBS buffer.

[0031] B8, ABPCs cultured from B7 were fed at 1.6 × 10⁻⁶. 6 One cell was seeded in a 75 cm² culture flask and cultured in complete medium containing 10% FBS with EVs removed for 48 hours.

[0032] B9 cells were cultured and their viability was assessed using the CCK-8 assay kit. Culture supernatant with cell viability exceeding 90% was collected.

[0033] B10: Centrifuge the supernatant collected from B9 at 750g for 20 minutes, then centrifuge at 2000g for 30 minutes; centrifuge the supernatant again at 16,000g for 70 minutes to remove the supernatant and obtain EVsABPCs. Resuspend the EVsABPCs in pre-cooled PBS and centrifuge again at 16,000g for 70 minutes to further purify the EVs.

[0034] B11, the passivated EVsABPCs were resuspended in 100µL of pre-cooled PBS, and the concentration of EVsABPCs was detected by NTA. They were then frozen and stored at -80℃.

[0035] Another technical solution adopted in this invention is the preparation method of antler-DCB@ABPCs-EVs bone graft material to obtain antler-DCB@ABPCs-EVs bone graft material.

[0036] The beneficial effects of this invention are:

[0037] The present invention relates to antler-DCB@EVsABPCs bone graft material and its preparation method, which injects EVs-ABPC composite hydrogel into Antler-DCB, thereby providing the graft material with a variety of bioactive molecules. At the same time, it exhibits a strong ability to recruit endogenous stem cells and endows them with phenotypes and molecular characteristics similar to ABPCs. This gives Antler-DCB a powerful ability to regulate osteogenic, angiogenesis, neurogenesis and immune response, thus providing a potential therapeutic intervention for the treatment of severe bone defects. Attached Figure Description

[0038] Figure 1 These are the manufacturing and characterization diagrams of DCBs for multiple species in Example 6;

[0039] Figure 2 This is a schematic diagram illustrating the physical and biological characteristics and functions of EVs in Example 6;

[0040] Figure 3 This is a schematic diagram of the construction of antler-inspired bone grafts - DCB@EVsABPCs in Example 6;

[0041] Figure 4 This is a schematic diagram of Example 6, showing how antler-DCB@EVsABPCs can rapidly induce bone formation in vivo;

[0042] Figure 5 This is a schematic diagram of angiogenesis stimulated by Antler-DCB@EVsABPCs in Example 6;

[0043] Figure 6 This is a schematic diagram illustrating the neurogenesis promotion of Antler-DCB@EVsABPCs in Example 6;

[0044] Figure 7 This is a schematic diagram of the inflammatory response during osteogenic process regulated by Antler-DCB@EVsABPCs in Example 6. Detailed Implementation

[0045] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings.

[0046] Example 1

[0047] This invention provides a method for preparing antler-DCB@EVsABPCs bone graft material, specifically implemented according to the following steps:

[0048] Step 1: Fabrication of the Antler-DCB scaffold;

[0049] Step 2: Prepare a hydrogel containing 10% HAMA and ABPCs-Evs;

[0050] Step 3: Mix equal volumes of hydrogel containing 10% HAMA and ABPCs-EVs. Each 1 ml of ABPCs-EVs contains 2.4 × 10⁻⁶ ppm of HAMA. 8 EVs.

[0051] Step 4: After mixing, shake at room temperature for 5 minutes and centrifuge at 4000 rpm for 5 minutes to remove air bubbles from the system. Then, immerse Antler-DCB in the HAMA hydrogel containing ABPCs-EVs for 30 minutes to ensure that the HAMA hydrogel is fully immersed in the pores of Antler-DCB.

[0052] Step 5: Remove the Antler-DCB hydrogel containing 10% HAMA and expose it to 405nm ultraviolet light for 30 seconds to obtain antler-DCB@ABPCs-EVs, and freeze at -80℃.

[0053] Example 2

[0054] Based on Example 1, step 1 specifically includes:

[0055] S1. By removing the outer skin, the cancellous bone of the naturally detached antler is obtained, and the cancellous bone of the antler is cut into suitable blocks as needed. In this embodiment, the cancellous bone of the antler is cut into 4×4mm bone samples, and the bone samples are repeatedly frozen and thawed, and then treated with 40kHz ultrasound for 20min.

[0056] S2, the bone sample after S1 ultrasonic treatment is immersed in ethanol and shaken on a shaker for 3 hours;

[0057] S3, Immerse the bone sample soaked in S2 in ether and shake on a shaker for 1.5 hours;

[0058] S4. The bone sample soaked in S3 was immersed in a 20 g / L sodium hydroxide solution and shaken on a shaker for 12 hours. After immersion, the bone sample was rinsed with running water for 3 hours. After rinsing, it was treated with 30% hydrogen peroxide for 24 hours. After treatment, it was rinsed again. The specific process of rinsing after treatment was as follows: the bone sample was rinsed with running water 3 times, 0.5 hours each time. Then, the bone sample was immersed in a DNase solution containing 1 mg / mL for 48 hours. After rinsing again, the sample was immersed in a solution containing 0.5 mg / mL RNase for 48 hours. The sample was rinsed with running water 3 times, 0.5 hours each time.

[0059] S5. After cleaning, the bone sample is placed in a mixture of chloroform and methanol at a volume ratio of 3:1 for 1 hour. The bone sample is then rinsed with distilled water and dried at 70°C for 1 day to produce Antler-DCB.

[0060] Example 3

[0061] Based on Example 2, the preparation of a hydrogel containing 10% HAMA is as follows:

[0062] The hydrogel containing 10% HAMA consists of HAMA, lithium phenyl phosphate (LAP), and deionized water in a ratio of 10:0.25:89.75.

[0063] A1. Prepare an initiator standard solution with a concentration of 0.25% by taking 10 mL of PBS buffer and adding it to a brown bottle containing 0.025 g of LAP. Heat the solution in a water bath at 40-50 °C for 15 minutes, shaking several times during the process, to obtain an initiator standard solution with a concentration of 0.25%.

[0064] A2 prepares a hydrogel containing 10% HAMA by: (1) taking the required mass of HAMA and placing it in a glass bottle or beaker; (2) taking the required volume concentration of 0.25% of the initiator standard solution and adding it to the above container, stirring and dissolving at room temperature for 0.5-1h; (3) heating the solution to 80℃ and maintaining it for 30 minutes; then quickly transferring it to an ice-water mixture and soaking for 5 minutes to obtain a hydrogel containing 10% HAMA.

[0065] Example 4

[0066] Based on Example 3, the preparation of ABPCs-Evs in step 2 is specifically as follows:

[0067] B1. Obtain the embryonic tissue 3-7 days after the hard antlers fall off. The 3-7 days after falling off ensure that the ABPCs in the tissue are in the proliferation stage. Cut open the skin covering the antler embryo to expose the underlying embryonic tissue.

[0068] B2, remove the germ tissue and cut it into 0.2 mm square pieces;

[0069] B3. Wash the cut tissue blocks three times with PBS buffer to remove blood and other components;

[0070] B4. Place the cleaned tissue blocks into a digestive solution (Type I: Type II: Type III collagenase = 1:1:1) and digest at 37°C for 30 minutes.

[0071] B5. The digested tissue was transferred to a 10 cm culture dish for primary cell culture. When the cell density reached 80%, flow cytometry was performed to separate the cells.

[0072] B6. The isolated cells were co-incubated with antibodies CD31, CD45, and CD235a, and then flow cytometry was performed to collect CD31-CD45-CD235a- cells to remove blood and immune-related cells, and the cells were cultured for a longer period.

[0073] B7. The collected CD31—CD45—CD235a— cells were digested, centrifuged, and co-incubated with ABPC marker antibodies CX43 and FGFR2. Flow cytometry was then performed to collect CX43+FGFR2+ cells, i.e., ABPCs, which were then cultured in DMEM complete medium containing 10% FBS buffer for EVs ABPC extraction.

[0074] B8, ABPCs cultured from B7 were fed at 1.6 × 10⁻⁶. 6 One cell was seeded in a 75 cm² culture flask and cultured for 48 hours in complete medium containing 10% FBS to remove EVs (to exclude interference from exosomes in serum).

[0075] B9, after culture, cell viability was assessed using the CCK-8 kit, and culture supernatant with cell viability exceeding 90% was collected to ensure the quality of the extracted EVsABPCs;

[0076] B10: Centrifuge the supernatant collected from B9 at 750g for 20 minutes, then centrifuge at 2000g for 30 minutes; centrifuge the supernatant again at 16,000g for 70 minutes to remove the supernatant. The resulting EVsABPCs are resuspended in pre-cooled PBS and centrifuged again at 16,000g for 70 minutes to further purify the EVs.

[0077] B11, the passivated EVsABPCs were resuspended in 100µL of pre-cooled PBS, and the concentration of EVsABPCs was detected by NTA. They were then frozen and stored at -80℃.

[0078] Example 5

[0079] This embodiment provides the method of using antler-DCB@EVsABPCs, specifically implemented according to the following steps: C1, 28 SD rats (approximately 300 grams, male) were used in the experiment, provided by the Experimental Animal Center of Air Force Medical University; C2, firstly, the rats were anesthetized with 3% (w / v) sodium pentobarbital; C3, a 3 cm incision was made on the right thigh of the rat; C4, the muscles and fascia were separated to expose the femur; C5, a trephine was used to create a 4 mm diameter and 4 mm depth defect in the middle of the femur; C6, the wound was cleaned and cooled with povidone-iodine; C7, a 4 mm diameter spherical scaffold was implanted into the defect and fixed to the femur with 5-0 absorbable sutures; C8, the muscles, aponeurosis, and skin were sutured layer by layer using 4-0 sutures; C9, to prevent infection, all rats were injected with 25,000 units of penicillin for the first three days after surgery.

[0080] The specific steps in the preparation method of the antler-DCB@EVs-ABPC bone graft material of the present invention are as follows: Step 1, S1, involves cutting the antler cancellous bone into 4×4mm blocks to facilitate further decellularization, thereby destroying immunogenic substances such as DNA by disrupting the cell structure. A freezing and thawing method is used, the principle of which is to disrupt the cell membrane by forming ice crystals, increasing the extractability of cell components. 40kHz ultrasound treatment is employed, the advantage of which is that ultrasound treatment can further disrupt the cell structure and promote the release of cell components.

[0081] The purpose of S2 is that ethanol helps remove lipids and other organic matter from the sample, while also helping to fix the tissue structure and reduce the impact of components on the matrix. The purpose of S3 is that ether soaking dissolves and removes lipid components in the cell membrane, and the volatile nature of ether effectively reduces the amount of solvent remaining in the tissue. The purpose of S4 is to dissolve organic components such as cell membranes and intracellular proteins, making it easier to remove cellular residues and helping to degrade residual cellular components in the sample, while eliminating potential microbial contamination. The purpose of the second washing is to degrade residual DNA to reduce the risk of immune responses, degrade residual RNA to reduce the risk of immune responses, and further remove fat and other lipid-soluble components from the bone tissue.

[0082] Step 2: The standard solution containing 0.25% initiator prepared in A1 can quickly induce crosslinking of HAMA under ultraviolet light, changing it from a liquid state to a gel state; EVsABPCs are mixed into the liquid HAMA prepared in A2, and the liquid HAMA solution needs to be in the pores of Antler-DCB, so the HAMA needs to be kept in a liquid state. After the three materials are mixed, crosslinking is carried out by ultraviolet light irradiation.

[0083] The present invention also provides antler-DCB@ABPCs-EVs bone graft material, which is composed of the following raw material components by weight percentage: Antler-DCB 16.3%~20.1%; 10% (W / V) HAMA 39.5%~43.7%; ABPC-EVs 40.1%~44.7%, wherein each 1 ml of ABPCs-EVs contains 2.4×10^8 EVs, 40.1%~44.7%.

[0084] The roles of each component in the antler-DCB@EVsABPCs bone graft material of this invention are as follows: Antler-DCB: Antler-DCB is a scaffold formed by decellularizing antler-DCB to obtain the ultrastructure, biophysical, and biochemical properties of the treated cancellous bone. Antler-DCB has the same crystal structure as hydroxyapatite in other vertebral DCBs. Antler-DCB has a significantly higher carbon content than other vertebral DCBs, existing in the form of CaCO3, which enhances the osteogenic activity of bone marrow mesenchymal stem cells (BMSCs) more effectively than hydroxyapatite. Furthermore, the Mg, S, and Si content in antler-DCB is 3.3 times higher than in other vertebral DCBs; these three elements are crucial for bone regeneration. Antler-DCB exhibits a stronger ability to promote new bone formation than other bone-DCBs, highlighting its potential as a natural bone repair graft material.

[0085] Methacrylamide hyaluronic acid (HAMA) at a mass concentration of 10 g / L has a wide range of important applications. First, it exhibits excellent biocompatibility. As a derivative of hyaluronic acid, HAMA inherits its good biocompatibility, meaning it can be safely used in vivo without causing significant immune or rejection reactions. Second, by modifying the methacrylic group on the hyaluronic acid molecular chain, it gains photocurability. This characteristic allows HAMA to form stable hydrogel structures through photocuring technology, providing a solid foundation for its application in the biomedical field. HAMA hydrogels can also serve as drug carriers. By controlling the structure and composition of the hydrogel, slow drug release can be achieved, thereby improving drug efficacy and reducing side effects.

[0086] Exosomes from antler bud progenitor cells (ABPCs-EVs): Antler bud progenitor cells are a novel type of stem cell for antler growth. The biological signals required for antler growth are transmitted through the extracellular vesicles (EVs) of ABPCs, including cytoplasmic proteins, lipids, and RNA. ABPC-EVs exhibit similar phenotypic morphology, epigenetic markers, and size distribution as BMSC-EVs. RNA transcriptomics and proteomics analyses show that upregulated genes in ABPC-EVs are mainly involved in cell cycle, cell motility, wound healing, and angiogenesis; downregulated genes are mainly involved in cell death and oxidative stress. Proteomics analysis indicates that upregulated proteins in ABPC-EVs are mainly involved in osteogenic, axonal growth, and blood circulation processes; downregulated proteins are mainly involved in inflammatory responses and oxidative stress. ABPC-EVs demonstrate a strong ability to recruit endogenous stem cells and endow them with phenotypes and molecular characteristics similar to ABPCs, which is beneficial for osteogenic, angiogenesis, neurogenesis, immune regulation, and thus promotes bone regeneration.

[0087] Example 6

[0088] This embodiment provides test results for the antler-DCB@EVs-ABPC bone graft material. The specific results are as follows: Figure 1 As shown, the bone structure within the antlers provides structural cues that promote rapid bone growth. Using antler-DCB (which preserves structural, biochemical, biophysical, and mechanical cues within the antlers)... Figure 1 (A and B). Deer antler-DCB exhibits a porous structure similar to that of the spine-DCB of deer, cattle, and humans. Figure 1 (C), but compared with different spinal-DCBs, its volumetric porosity (87.60±5.45%), pore size (626.5±63.9 mm), and specific surface area (199.95±16.48 m² / g) were significantly higher. Figure 1 The large porous structure may promote bone ingrowth and osseointegration of antler-DCB with surrounding bone. Furthermore, the surface roughness and hydrophilicity of antler-DCB are significantly higher than those of different vertebral-DCBs (D to F). Figure 1 (G and H). This promotes cell adhesion and fluid infiltration, which contributes to bone regeneration. Mechanical properties are a key element supporting regenerative bone grafts. The maximum stress of staghorn DCB falls within the mechanical range of cancellous bone, indicating its mechanical suitability for bone regeneration. Figure 1 X-ray diffraction (XRD) analysis showed that staghorn-DCB had characteristic peaks of hydroxyapatite, and peaks were also observed in other DCBs. Figure 1 J), indicating that hydroxyapatite (Ca 10[PO4]6[OH]2) is the main component of antler-DCB and spinal-DCB. Furthermore, energy-dispersive spectroscopy revealed that the carbon content of antler-DCB is significantly higher than that of different spinal-DCBs. Figure 1 Fourier transform infrared spectroscopy suggests that carbon may be found in the form of CaCO3. Figure 1 Furthermore, the Mg, S, and Si contents in antler-DCB were more than 3.3 times higher than those in different spinal cord-DCBs. Figure 1 M to O). These three elements are crucial for bone regeneration. DCB was implanted into femoral defects in rats to further evaluate its osteogenic effect in vivo. At 12 weeks post-implantation, the percentage of new bone formation in the antler-DCB group was significantly higher than that in the deer, bovine, and human-DCB groups ( Figure 1 (P). In the antler DCB group, new bone formation in the locally defected areas showed significantly higher bone mineral density (BMD), bone volume / total tissue volume (BV / TV), and trabecular thickness (Tb.Th.) at 12 weeks. Figure 1 (Q to T). These findings indicate that antler-DCB has a stronger ability to promote new bone formation than commercially available DCB, highlighting its potential as a natural graft material for bone repair. Figure 1 (A) Schematic diagram of the manufacturing process and characterization techniques of decellularized bone marrow. (B) Naturally shed sika deer antlers. (C) Scanning electron microscope (SEM) image of the DCB surface. (D to F) Measurements of porosity (D), pore size (E), and specific surface area (SSA) (F) in DCB (n=4). (G) Measurement of roughness (Ra) of DCB using 3D atomic force microscopy (AFM). (H) Measurement of water contact angle (WCA) in DCB (n=4). (I) Compressive stress-strain curve of DCB. (J) X-ray diffraction (XRD) pattern of DCB. (K) Fourier transform infrared (FT-IR) spectrum of DCB. (From left to right) Quantitative analysis of C (L), S (M), mg (N), and Si (O) contents in each group (n=4). (P) Hematoxylin-eosin (H&E) and Masson staining images show new bone formation (NB, newly formed bone tissue) at 12 weeks postoperatively. (Q) 3D reconstruction and 2D micro-CT images show regenerated bone around the defect. (Right to right) Quantitative analysis of microstructural parameters of regenerated bone tissue, including BMD(R), BV / TV(S), and Tb.Th(T) (n=5). All statistics are expressed as mean ± SD. Statistical analysis was performed using ANOVA and Bonferroni post-hoc tests. *P<0.05, **P<0.01, ***P<0.001.

[0089] like Figure 2As shown, bioactive substances within growing antlers are inevitably lost during decellularization. Therefore, it is proposed to supplement antler-DCB with EVsABPCs to enhance its bone regeneration-related bioactive cues. Their characteristics were characterized and compared with those of EVs in the most widely used BMSCs (EVsBMSCs). Figure 2 EVsABPCs exhibit similar morphology, surface markings, and size distribution to EVsBMSCs. Figure 2 B and C). Both EVsABPCs and EVsBMSCs were positive for CD81, CD63, and TSG101, which are typical markers of EVs ( Figure 2 D). Then, RNA transcriptomics and proteomics analyses were performed to characterize their composition ( Figure 2 (E to I). Overall, 658 differentially expressed genes (DEGs) were identified between EVsABPCs and EVsBMSCs. Figure 2 (F) and 370 differentially expressed proteins (DEPs) Figure 2 Enrichment analysis revealed 282 upregulated genes in EVsABPCs, primarily involved in the cell cycle (e.g., G). Hspa8, Kpnb1, Smc4 ), cell movement (e.g., Cfl1, Fgf7, Sema4g ), wound healing (e.g., Actg1, Ano6 Anxa2 ) and angiogenesis (e.g., Dcn, Cd63, Tcf4 ), while 376 downregulated genes are mainly involved in cell death (e.g., Dynll1, Chmp2b, Hsp90aa1 ) and oxidative stress (e.g., Ptpn11, Hspa8, Arpc2 () Figure 2 In proteomics analysis, 227 upregulated proteins in EVsABPCs were mainly involved in osteogenic processes (e.g., FOSL2, GLG1, ANXA2, RUNX2, THBS3), axonal growth (e.g., CACNA1C, STMN1, OLIG1, DCLK1), and blood circulation processes (e.g., CD36, PDE3A, TTN). 143 downregulated proteins were associated with inflammatory responses (e.g., HP, ORM1, F12) and oxidative stress (e.g., PDGFRB, APOA4, PPIA, SLCLA1). Figure 2 (H and I). In summary, EVsABPCs contain stronger bioactive signals that promote tissue regeneration than EVsBMSCs, highlighting their therapeutic potential in bone injury repair. Secondly, the chemotactic effect of EVsABPCs on BMSC migration was verified. Figure 2 In scratch testing, EVsABPCs improved wound size recovery by 4.2 times, while EVsBMSCs improved recovery by 1.8 times compared to PBS. Figure 2 K and M). In transwell migration assays, the number of migrating cells observed in the EVsABPCs group was 3.5 times that of the vector group and 1.9 times that of the EVsBMSCs group. Figure 2 These findings indicate that EVsABPCs have a stronger chemotactic effect on BMSCs than EVsBMSCs. Further transcriptomic analysis revealed 416 DEGs (, L and N) between BMSCs treated with EVsABPCs and EVsBMSCs. Figure 2 (O to Q). These genes are involved not only in cell movement but also in the cell cycle, bone mineralization, neurogenesis, and stem cell proliferation. These findings suggest that EVsABPCs can induce a phenotypic shift in BMSCs that favors tissue regeneration. Figure 2 In the middle, (A) Schematic diagram of EV characterization techniques. (B to D) Characterization of EVs by (B) Transmission Electron Microscopy (TEM), (C) Nanoparticle Tracking Analysis (NTA), and (D) Western Blotting. (E) Heatmap showing the distribution of differentially expressed genes (DEGs) between EVsABPCs and EVsBMSCs, and the enrichment pathways of these DEGs (right). (F) Volcano plot of DEGs in EVsABPCs and EVsBMSCs. Red dots indicate significant upregulation in EVsABPCs; blue dots indicate significant upregulation in EVsBMSCs; gray dots indicate no significant upregulation. (G) Volcano plot of differentially expressed proteins (DEPs) in EVsABPCs and EVsBMSCs. (H and I) Bar plots showing the enriched gene ontology (GO) terms for upregulated genes (H) and downregulated genes (I) in EVsABPCs and EVsBMSCs. (J) Workflow for assessing the effects of EVs on BMSCs. (K and M) Wound healing rate of BMSCs after each treatment within 24 hours (n=4). (L and N) BMSC migration behavior analyzed using transwell analysis (n=4). (O) Multiple volcano plots showing the DEG of BMSCs treated with PBS, EVsABPCs, and EVsBMSCs. (P) Principal component analysis illustrating differences in BMSCs treated with PBS, EVsBMSCs, and EVsABPCs. (Q) Network plots showing enriched GO terms and pathways of upregulated genes in the EVsABPCs and EVsBMSCs groups. All statistics are expressed as mean ± SD. Analysis was performed using ANOVA and Bonferroni post-hoc test. ***P < 0.001.

[0090] like Figure 3As shown, antler-inspired bone grafts (antler-DCB@EVsABPCs) were constructed by integrating EVsABPCs into antler-DCB. Given that exogenous EVs are rapidly eliminated after local application, a controlled release system was established to maintain their long-term effective concentration for bone regeneration. EVs were encapsulated in a hyaluronic acid methacryloyl (HAMA) hydrogel, which binds to the key surface marker versican (VCAN) of EVs to achieve their gradual release. Figure 3 , A). EVs are uniformly distributed in the hydrogel, as indicated by PKH26 labeling ( Figure 3 Secondly, a series of computational techniques were used to evaluate the detailed modes of interaction between HAMA and VCAN. The results show that VCAN / HAMA has good binding stability (B). Figure 3 (C and D). Furthermore, radius of gyration (Rg) analysis showed that the combination of HAMA and VCAN had almost no effect on the changes in its density and stiffness over time. Figure 3 E and F). The Gibbs free energy landscape was then calculated, and the free energy value of the VCAN / HAMA complex (<7.04 kJ / mol) was lower than that of VCAN (<7.64 kJ / mol), indicating that the VCAN / HAMA complex exhibits overall thermodynamic stability ( Figure 3 The distribution of hydrogen bonds (H-bonds) between VCAN and HAMA further confirms the stable bonding between HAMA and the EV surface. Figure 3 Furthermore, the negatively charged HAMA hydrogel forms a strong interfacial electronic interaction with the positively charged staghorn-DCB (H). Figure 3 , I and J), and endowed the antler-DCB biphase sponge with a structure and excellent pore size distribution ( Figure 3 K and L). Notably, in situ fluorescence signals of hydrogels containing cyanine 5.5 monoacids were observed at different time points after in vivo implantation. Under in vivo conditions, HAMA hydrogels in staghorn-DCB@EVsABPCs exhibited slower degradation kinetics than HAMA hydrogels alone. Figure 3 M and N), which may be due to the extensive amide bonds formed between HAMA and the minerals in antler-DCB. Furthermore, EVs are continuously released from the antler-DCB@EVsABPCs composite for at least 18 days. Figure 3 In addition, antler-DCB@EVsABPCs exhibit excellent hydrophilicity (O). Figure 3 (P) and good biocompatibility ( Figure 3 (Q), making it a potential candidate material for bone repair applications. Figure 3(A) Schematic diagram of the interaction between HAMA hydrogel and EVs. (B) Examination of PKH26 (red) labeled EVs in HAMA hydrogel by z-stack scanning using confocal laser scanning microscopy. Fluorescence intensity results (left) are shown as a 3D view of surface remodeling, presented in angled (middle) and frontal (right) views. (C-F) Root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), and solvent accessible surface area (SASA) of VCAN and VCAN / HAMA. (G) Free energy landscape of VCAN and VCAN / HAMA hydrogels based on principal coordinate analysis. (H) Docking simulation of VCAN and HAMA hydrogel compounds. (I) Schematic diagram of the fabrication and characterization of antler-DCB@EVs. (J) Quantitative analysis of the surface potential of antler-DCB and HAMA hydrogels by Kelvin probe force microscopy. (K) Pore size distribution of antler-DCB@EVs ABPCs. (L) Characterization of antler-DCB@EVsABPCs by TEM. (M and N) Degradation rates of antler-DCB@EVsABPCs and HAMA hydrogels (n=3). (O) Cumulative EV release was determined at days 1, 3, 6, 9, 12, 15, and 18 (n=3) using the dioctanine assay. (P) WCA measurements of antler-DCB@EVsABPCs. (Q) CCK-8 analysis of BMSCs cultured on different scaffolds for days 1, 3, and 7 (n=3). All statistics are expressed as mean ± SD. Statistical analysis was performed using ANOVA and Bonferroni post-hoc tests. *P<0.05, **P<0.01, and ***P<0.001.

[0091] like Figure 4 As shown, a rat femoral condyle defect model was established to evaluate the osteogenic activity of staghorn-DCB@EVsABPCs in vivo. Figure 4 A). At the end of eight weeks, the untreated group showed little bone growth and deformities due to a lack of effective structural support. Figure 4 The figure (C) indicates that the size of the femoral defect exceeds the critical size for spontaneous healing. More new bone was observed in rats implanted with antler-DCB than in untreated rats, which may be attributed to biochemical and biophysical cues in antler-DCB. Notably, introducing bioactive EVs into antler-DCB further enhanced its osteogenic potential, with a greater effect observed in the antler-DCB@EVsABPCs group. Figure 4 C). The BMD, Tb.Th., Tb.N. and BV / TV values ​​of new bone in the antler DCB@EVsABPCs group were significantly higher than those in other groups. Figure 4In summary, antler-DCB@EVsABPCs effectively triggered new bone formation in vivo. Histological analysis further confirmed the strong osteogenic capacity of antler-DCB@EVsABPCs. Only a small amount of new bone tissue was observed after 8 weeks. Figure 4 B). The antler-DCB group and the antler-DCB@EVs group showed more new bone formation, with the antler-DCB@EVsABPCs group exhibiting the most new bone tissue. Figure 4 B). Sequential fluorescence labeling was used to quantify the mineralization rate of new bone. The mineral deposition rate (MAR) of the antler-DCB@EVsABPCs group was 1.6 times higher than that of the antler-DCB@EVsBMSCs group and 2.3 times higher than that of the antler-DCB group. Figure 4 Furthermore, the expression of osteogenic markers (ALP, RUNX2, and OCN) was highest in the antler-DCB@EVsABPCs group, followed by the antler-DCB@EVsBMSCs group and the antler-DCB group (F). Figure 4 The results showed that antler-DCB@EVsABPCs promoted bone formation in vivo at both the histological and molecular levels. Interestingly, at 2 weeks post-surgery, the number of BMSCs (CD45–DC44+CD29+) detected in the bone defect area was significantly higher in the antler-DCB@EVsABPCs group than in other groups. Figure 4 These findings indicate that antler-DCB@EVsABPCs recruit more BMSCs to the injury site. Regenerated tissues from each group were collected 2 weeks post-infection and RNA-seq analysis was performed. Compared to the antler-DCB@EVsBMSCs and antler-DCB groups, 1,405 and 879 DEGs were observed in the regenerated tissues of the antler-DCB@EVsABPCs group, respectively (Log2|FC|>1, P<0.5). Subsequent cluster analysis and GO analysis of the combined DEGs among the three groups identified five clusters with different key characteristics and biological significance (H and I). Figure 4Cluster 1 contained genes specifically enriched in the antler-DCB@EVsABPCs group, which are involved in ossification, angiogenesis, and neurogenesis. Cluster 5 contained genes specifically downregulated in both antler-DCB@EVsABPCs and antler-DCB@EVsBMSCs, which are primarily involved in regulating immune and inflammatory responses. Transcriptome profiles of regenerated tissues from the antler-DCB@EVsABPCs group were compared with those from antler growth centers, identifying 430 shared upregulated genes enriched in pathways crucial for rapid antler development, including those associated with the mitogen-activated protein kinase (Erk1 / 2) signaling pathway, bone morphogenetic protein (BMP) signaling pathway, transforming growth factor-β (TGF-β) signaling pathway, and WNT signaling pathway. Notably, marker genes for ABPCs (such as Prrx1, Tnc, Dlx5, Ptn, and Sox4) were also found in the antler-DCB@EVsABPCs group, indicating that antler-DCB@EVsABPCs triggers a bone growth process similar to antler growth. Figure 4 In summary, these results indicate that antler-DCB@EVsABPCs trigger transcriptomic profiles at localized damage sites, partially mimicking the molecular dynamics of antler growth centers. Figure 4 (A) Schematic diagram exploring the osteogenic capacity of antler-DCB@EVsABPCs. (B) H&E and Masson stained images showing new bone formation at 8 weeks post-surgery (M, material; NB, new bone tissue). (C) 2D micro-CT (top) and 3D reconstruction (bottom) images showing regenerated bone around the defect. (D) Quantitative analysis of microstructural parameters of regenerated bone tissue, including BV / TV, BMD, Tb.Th., and Tb.N. (n=5). (E and F) Representative images and summed quantifications showing newly formed bone trabeculae labeled with calcein AM (green fluorescence) and alizarin red (red fluorescence) at 2 and 4 weeks post-scaffold insertion (n=5). (G) Representative Western blot images showing ALP, RUNX2, and OCN levels in new bone tissue with various scaffolds. (H and I) Flow cytometry detection of recruitment of various scaffolds on BMSCs in vivo (n=5). (J) Cluster analysis and GO analysis of DEG in different groups identified key distinguishing features and biological significance. (K) Schematic diagram of the common mechanism of antler growth and bone growth induced by antler-DCB@EVsABPCs. All statistics are expressed as mean ± SD. Statistical analysis was performed using ANOVA with Bonferroni post-hoc test. **P < 0.01 and ***P < 0.001.

[0092] like Figure 5As shown, rapid antler growth is accompanied by rapid vasodilation. Consistent with this, proteins related to angiogenesis are significantly enriched in EVsABPCs ( Figure 5 (A to C). Therefore, we evaluated the effect of staghorn-DCB@EVsABPCs on angiogenesis in regenerated bone. PKH26-labeled EVs were internalized and distributed in the perinuclear region of human umbilical vein endothelial cells (HUVECs). Figure 5 (D to E), highlighting their potential impact on target cells. After 24 hours of treatment with staghorn-DCB@EVsABPCs, the migration ability of HUVECs was significantly improved, and the number of transmembrane cells was significantly higher than that in the staghorn-DCB@EVsBMSCs treatment group (D to E). Figure 5 (F and H). Furthermore, compared to the staghorn-DCB@EVsBMSCs group, more mature tubular structures and higher density of cell connections were observed in the staghorn-DCB@EVsABPCs group (F and H). Figure 5 (G and I). These findings indicate that antler-DCB@EVsABPCs promote HUVEC migration and angiogenesis more effectively than antler-DCB@EVsBMSCs. We further evaluated the effects of EVsABPCs on angiogenesis in vivo ( Figure 5 , J). Eight weeks after vaccination, the vascular structure improvement was most significant in the antler-DCB@EVsABPCs group, followed by the antler-DCB@EVsBMSCs group and the antler-DCB group, while the improvement was least significant in the untreated group. Figure 5 , K). Eight weeks after inoculation, the vascular area of ​​the antler-DCB@EVsABPCs group was 1.9 times that of the antler-DCB@EVsBMSCs group and 2.4 times that of the antler-DCB group. Figure 5 In addition, the expression of two representative vascular markers, platelet endothelial cell adhesion molecule-1 (CD31) and endoheptin (EMCN), was significantly higher in the staghorn-DCB@EVsABPCs group than in other groups. Figure 5 (M to O). In summary, these results indicate that antler-DCB@EVsABPCs exhibit a strong pro-angiogenic capacity, promoting neovascularization to support extensive new bone formation. Figure 5In the middle, (A to C) protein sequencing analysis of DEPs related to angiogenesis regulation in EVsABPCs and EVsBMSCs. (D and E) Cell uptake assays showing PKH26-labeled EVs internalized and distributed in HUVECs. HUVECs were stained with phalloidin (green), and EVs with PKH26 (red). (F and H) Transwell analysis of HUVEC migration behavior (n=3). (G and I) Tube-forming capacity of HUVECs under each treatment method (n=3). (J) Schematic diagram exploring the angiogenic capacity of staghorn-DCB@EVsABPCs. (K and L) 3D reconstructed images and summary quantitative data showing newly formed vessels at 4 and 8 weeks post-implantation in different groups (n=5). (M) Representative immunofluorescence images of CD31 (green), EMCN (green), and nuclei (blue) staining at eight weeks. (N and O) Quantitative analysis of all positively stained areas. All statistics are presented as mean ± SD. Statistical analysis was performed using ANOVA and Bonferroni's post-thermal method. **P<0.01, ***P<0.001.

[0093] like Figure 6 As shown, neurogenic proteins such as ATP2B2, IGFBP5, GIT1, and SMG9 were significantly enriched in EVsABPCs. Figure 6 (A to C). Therefore, the potential impact of EVsABPCs on neuronal regeneration capacity was investigated. PKH26-labeled EVs were co-localized in the cell body and axon of neurons ( Figure 6 (D and E) indicates that EVsABPCs and EVsBMSCs can be directly internalized by neurons. In the EVsABPCs treatment group, the axon length of neurons (428.5±22.7μm) was significantly higher than that of the EVsBMSCs (363.0±42.9μm) and carrier (258.3±18.3μm) groups. Figure 6 F, G, and I). A similar trend was also observed in dorsal root ganglion (DRG) explants. Figure 6 The neurite area and the average length of the five longest axons in the EVsABPCs group were significantly greater than those in the EVsBMSCs group (H). Figure 6 These findings demonstrate that EVsABPCs possess a strong ability to promote axonal growth. Subsequently, sensory nerve fibers were visualized by labeling with calcitonin gene-related peptide (CGRP), a marker of sensory nerves within newly formed bone. Compared to the remaining groups, antler-DCB@EVsABPCs exhibited the highest CGRP expression (J). Figure 6(K to M). This finding suggests that antler-DCB@EVsABPCs achieve greater sensory innervation, which may indirectly contribute to their beneficial effects on bone growth. The conversion of nerve fibers from sympathetic to cholinergic nerves is beneficial to bone cell survival. The conversion of sympathetic to cholinergic nerves in the antler-DCB@EVsABPCs group was found to be significantly higher than in other groups, manifested by increased expression of the cholinergic marker vesicular acetylcholine transporter (VAChT) and decreased expression of the norepinephrine marker tyrosine hydroxylase (TH). Figure 6 (N to P). Furthermore, acetylcholine levels were highest in the antler-DCB@EVsABPCs group, followed by the antler-DCB@EVsBMSCs group and the antler-DCB group, and lowest in the untreated group. Figure 6 (Q). This study confirmed the beneficial role of antler-DCB@EVsABPCs in the sympathetic-cholinergic conversion process during new bone formation. Figure 6 (A to C) Protein sequencing analysis of DEPs related to neurogenesis regulation in EVsABPCs and EVsBMSCs. (D and E) Cell uptake assays show that PKH26-labeled EVs are internalized and distributed in neurons. Neurons are stained with Tuj1 (green), cell nuclei are stained with DAPI (blue), and EVs are stained with PKH26 (red). (F to H) DRG neurons and explants are co-cultured with different groups of EVs for 72 hours. (I) Quantification of axonal length of DRGs (n=4). (J) Ratio of total neurite area to total explant area (n=4). (K) Schematic diagram showing that antler-DCB@EVsABPCs promote neurogenesis and the conversion of neuronal fibers from sympathetic to cholinergic nerves. (L and M) Immunofluorescence staining shows the distribution of CGRP+ neurons in the bone regeneration area after different treatments (n=5). (N) Representative immunofluorescence images of vesicular acetylcholine transporter (VAChT) (green), sympathetic marker tyrosine hydroxylase (TH) (green), and cell nucleus (blue) stained at rat bone formation sites. (O and P) Quantitative analysis of all positively stained regions in cholinergic and sympathetic nervous systems (n=5). (Q) Acetylcholine content in different groups (n=5). All statistics are expressed as mean ± SD. Statistical analysis was performed using ANOVA with Bonferroni post-hoc test. *P<0.05, **P<0.01, and ***P<0.001.

[0094] like Figure 7 As shown, compared with EVsBMSCs, many proteins related to the inflammatory response (such as ORM1, CTSC, COL6A1, and FABP4) were downregulated in EVsABPCs. Figure 7(A to C). Therefore, the effect of EVsABPCs on macrophage phenotypic transformation was investigated. Compared with EVsBMSCs and PBS-induced macrophages, EVsABPCs treatment led to a significant decrease in iNOS (M1 marker) expression and a significant increase in Arg1 (M2 marker) expression in macrophages. Figure 7 Furthermore, EVsABPCs increased the level of anti-inflammatory IL-10 and decreased the levels of pro-inflammatory factors TNF-α, PGE2, and IL-1β in BMSCs (D and E). Figure 7 The results showed that EVsABPCs could induce M1 to M2 macrophage polarization and reduce the inflammatory response, which may contribute in part to their potent beneficial effects on bone regeneration. Next, the in vivo immunomodulatory capacity of antler-DCB@EVsABPCs was evaluated within 7 days post-implantation. Figure 7 (G). The area of ​​iNOS+ cells (M1 macrophages) was lowest in the antler-DCB@EVsABPCs group, followed by the antler-DCB@EVsBMSCs and antler-DCB groups, with the lowest area in the untreated group. The area of ​​CD206+ cells (M2 phenotype) showed the opposite trend, with the largest area in the antler-DCB@EVsABPCs group (G). Figure 7 These findings confirm that staghorn-DCB@EVsABPCs can convert M1 macrophages to the M2 phenotype in vivo. Furthermore, compared to staghorn-DCB@EVsBMSCs treatment, staghorn-DCB@EVsABPCs treatment significantly increased the expression of the anti-inflammatory factor Arg1 and decreased the levels of pro-inflammatory factors (H and I). Figure 7 J and K), which are crucial for bone growth. Figure 7 (A-C) Sequencing analysis of DEP proteins associated with inflammation regulation in EVsABPCs and EVsBMSCs. (D) Quantification of iNOS+F4 / 80+ cell rate. (E) Quantification of Arg1+F4 / 80+ cell rate. (F) Assessment of pro-inflammatory markers TNF-α, PGE2, IL-10, and IL-1β in macrophages by enzyme-linked immunosorbent assay (ELISA). (G) Schematic diagram of in vivo immune regulation in staghorn-DCB@EVsABPCs within 7 days post-implantation. (H) Representative immunofluorescence images of iNOS (red), CD206 (green), and nuclei (blue) staining. (I) Quantitative analysis of all positively stained regions (n=4). (J and K) Western blot analysis and pooled quantitative data of TNF-α, COX2, and Arg1. All statistics are expressed as mean ± SD. Statistical analysis was performed using ANOVA and Bonferroni post-hoc test. *P<0.05, **P<0.01 and ***P<0.001.

Claims

1. A method for preparing antler-DCB@EVsABPCs bone graft material, characterized in that, The specific steps are as follows: Step 1: Fabrication of the Antler-DCB scaffold; Step 2: Prepare a 10% HAMA hydrogel with ABPCs-Evs; Step 3: Take an equal volume of hydrogel containing 10% HAMA and mix it with ABPCs-EVs; Step 4: Immerse Antler-DCB in a 10% HAMA hydrogel containing ABPCs-EVs for 30 minutes; Step 5: Remove the Antler-DCB hydrogel containing 10% HAMA and expose it to 405nm ultraviolet light for 30 seconds to obtain antler-DCB@ABPCs-EVs bone graft material, and freeze it at -80℃.

2. The method for preparing the antler-DCB@EVsABPCs bone graft material as described in claim 1, characterized in that, The specific steps of step 1 are as follows: S1, cut the cancellous bone of the antler into 4×4mm bone samples, repeatedly freeze and thaw, and then treat with ultrasound; S2, the bone sample after S1 ultrasonic treatment is immersed in ethanol and shaken on a shaker for 3 hours; S3, Immerse the bone sample soaked in S2 in ether and shake on a shaker for 1.5 hours; S4. The bone sample soaked in S3 was immersed in a sodium hydroxide solution with a mass concentration of 20 g / L and shaken on a shaker for 12 hours. After soaking, the bone sample was washed with running water for 3 hours. After washing, it was treated with hydrogen peroxide with a mass fraction of 30% for 24 hours. After treatment, it was washed again. S5. After cleaning, the bone sample is placed in a mixture of chloroform and methanol at a volume ratio of 3:1 for 1 hour. The bone sample is then rinsed with distilled water and dried at 70°C for 1 day to produce Antler-DCB.

3. The method for preparing the antler-DCB@EVsABPCs bone graft material as described in claim 2, characterized in that, The ultrasonic treatment in S1 specifically involves treating the bone sample with 40kHz ultrasound for 20 minutes.

4. The method for preparing the antler-DCB@EVsABPCs bone graft material as described in claim 2, characterized in that, The specific steps for the second cleaning in S4 are as follows: wash the bone sample three times with running water for 0.5 hours each time, then soak the bone sample in a DNase solution containing 1 mg / mL for 48 hours, and after the second cleaning, soak the sample in a solution containing 0.5 mg / mL RNase for 48 hours, and wash it three times with running water for 0.5 hours each time.

5. The method for preparing the antler-DCB@EVsABPCs bone graft material as described in claim 1, characterized in that, The specific steps for preparing HAMA hydrogel in step 2 are as follows: A1. Take 10 mL of PBS buffer and add it to a bottle containing 0.025 g of LAP. Heat in a water bath at 40-50 °C for 15 minutes, shaking several times during the process, to obtain the initiator standard solution. A2. Take the required mass of methacrylamide hyaluronic acid and put it into a container; take the required volume of initiator standard solution and add it to the container containing methacrylamide hyaluronic acid. Stir and dissolve at room temperature for 0.5-1 h, then heat the solution to 80°C for 30 minutes; then transfer it to an ice-water mixture and soak for 5 min to obtain HAMA hydrogel.

6. The method for preparing the antler-DCB@EVsABPCs bone graft material as described in claim 5, characterized in that, The HAMA hydrogel described in A2 comprises HAMA, lithium phenyl phosphate, and deionized water, wherein the volume ratio of HAMA, lithium phenyl phosphate, and deionized water is 10:0.25:89.

75.

7. The method for preparing the antler-DCB@EVsABPCs bone graft material as described in claim 1, characterized in that, The specific steps for preparing ABPCs-Evs in step 2 are as follows: B1, obtain embryonic tissue 3-7 days after the antlers fall off; B2, cut the germ tissue into 0.2 mm square pieces; B3. Wash the cut tissue blocks three times with PBS buffer. B4. Place the cleaned tissue blocks into a digestion solution with a volume ratio of type I: type II: type III collagenase of 1:1:1 and digest at 37°C for 30 minutes. B5. The digested tissue block was transferred to a 10 cm culture dish for primary cell culture. When the cell density reached 80%, flow cytometry was performed to separate the cells. B6. Cells separated from B5 were co-incubated with antibodies CD31, CD45, and CD235a, and then flow cytometry was performed to collect cells containing CD31-CD45-CD235a-. B7. Collect cells containing CD31-CD45-CD235a- from B6, digest and centrifuge them, incubate the separated cells with ABPC marker antibodies CX43 and FGFR2, and perform flow cytometry sorting to collect ABPCs. Then, culture them in DMEM complete medium containing 10% FBS buffer. B8, ABPCs cultured from B7 were fed at 1.6 × 10⁻⁶. 6 One cell was seeded in a 75 cm² culture flask and cultured in complete medium containing 10% FBS with EVs removed for 48 hours. B9 cells were cultured and their viability was assessed using the CCK-8 assay kit. Culture supernatant with cell viability exceeding 90% was collected. B10: Centrifuge the supernatant collected from B9 at 750g for 20 minutes, then centrifuge at 2000g for 30 minutes; centrifuge the supernatant again at 16,000g for 70 minutes to remove the supernatant and obtain EVsABPCs. Resuspend the EVsABPCs in pre-cooled PBS and centrifuge again at 16,000g for 70 minutes to further purify the EVs. B11, the purified EVsABPCs were resuspended in 100µL of pre-chilled PBS, and the concentration of EVsABPCs was detected by NTA. The EVsABPCs were then frozen and stored at -80℃.

8. The antler-DCB@ABPCs-EVs bone graft material prepared by the method described in any one of claims 1-7.

Images