Antler-DCB-coated EVsABPCs bone graft material and preparation method thereof

Abstract

The invention discloses a preparation method of an antler-DCB (at) EVsABPCs bone graft material. The preparation method specifically comprises the following steps: step 1, preparing an Antler-DCB scaffold; step 2, preparing hydrogel containing 10% of HAMA and ABPCs-Evs (2, 2 '-(1, 2, 4-trimethyl-1, 3, 4- Step 3, mixing; step 4, taking Antler-DCB, and immersing the Antler-DCB into the HAMA hydrogel containing the ABPCs-EVs; and step 5, exposing to 405nm ultraviolet light for 30 seconds to obtain the bone graft material. The invention further discloses the antler-DCB (at) EVsABPCs bone graft material. According to the antler-DCB-coated EVsABPCs bone graft material and the preparation method thereof disclosed by the invention, the strong capability of adjusting osteogenesis, angiogenesis, neurogenesis and immune response is obtained, so that a potential therapeutic intervention measure is provided for treating serious bone defects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical material preparation, and specifically relates to a method for preparing a antler-DCB@EVsABPCs bone transplant material, and also relates to a antler-DCB@EVsABPCs bone transplant material. Background Art

[0002] Bone injury is one of the most common disabling injuries worldwide, with more than 400 million patients suffering from acute or long-term consequences of such injuries, which seriously affects the quality of life of individuals. Bone regeneration is not an independent bone growth process, but a complex and dynamic process involving the coordinated growth of bone, vascular and neural structures, complicated by a vigorous immune response. In the past decade, significant progress has been made in the development of various bone transplants by integrating cells, growth factors and biomaterials. However, the current clinical integration rate of engineered bone transplants is still low, and the lack of a disposable bone transplant construction strategy has led to unresolved challenges in achieving effective bone regeneration. Existing biomedical materials cannot achieve the practical requirements of low immunogenicity, high safety, high repair rate and high bioactivity. Summary of the invention

[0003] The purpose of the present invention is to provide a method for preparing antler-DCB@EVsABPCs bone transplant materials, which solves the problems of immune rejection and insufficient biological activity in existing natural bone transplants.

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

[0005] The first technical solution adopted by the present invention is a method for preparing antler-DCB@EVsABPCs bone transplant material, which is specifically implemented according to the following steps: Step 1, preparing the Antler-DCB stent; Step 2, preparing 10% HAMA hydrogel and ABPCs-Evs; Step 3, taking equal volumes of hydrogel containing 10% HAMA and mixing them with ABPCs-EVs; Step 4, take Antler-DCB and immerse it in 10% HAMA hydrogel containing ABPCs-EVs for 30 minutes; Step 5, take out the Antler-DCB containing 10% HAMA hydrogel and expose it to 405nm ultraviolet light for 30 seconds to obtain DCB@ABPCs-EVs bone transplant material, which is frozen and stored at -80°C.

[0006] The first technical solution of the present invention is also characterized in that step 1 specifically comprises the following steps: S1, antler cancellous bone was cut into 4 × 4 mm bone samples and then treated with ultrasound after repeated freezing and thawing; S2, the bone samples treated with ultrasound in S1 were immersed in ethanol and shaken on a shaker for 3 h; S3, soak the bone sample soaked in S2 in ether and shake it on a shaker for 1.5 hours; S4, soaking the bone sample soaked in S3 in a sodium hydroxide solution with a mass concentration of 20 g / L and shaking on a shaker for 12 hours, washing the bone sample with running water for 3 hours after soaking, treating it with 30% hydrogen peroxide for 24 hours after washing, and washing it again after treatment; S5, the cleaned bone sample was placed in a mixture of chloroform and methanol in a volume ratio of 3:1 for 1 hour, and then the bone sample was rinsed with distilled water and dried at 70°C for 1 day to produce Antler-DCB.

[0007] The specific ultrasonic treatment in S1 is: using 40kHz ultrasonic waves to treat the bone sample for 20 minutes.

[0008] The re-cleaning in S4 is as follows: the bone sample is washed with running water for 3 times, each time for 0.5 hours, and then the bone sample is immersed in a solution containing 1 mg / mL DNase for 48 hours. After washing again, the sample is immersed in a solution containing 0.5 mg / mL RNase for 48 hours, and washed with running water for 3 times, each time for 0.5 hours.

[0009] The specific steps for preparing HAMA hydrogel in step 2 are as follows: A1, take 10mL PBS buffer, add it to the bottle containing 0.025g LAP, heat it in a 40-50℃ water bath to dissolve it for 15 minutes, shake it several times during the process, and prepare the initiator standard solution. A2, take the required mass of methacryloyl hyaluronic acid and put it into a container; take the required volume of the initiator standard solution and add it to the container containing the methacryloyl hyaluronic acid, stir and dissolve it at room temperature for 0.5-1h, then heat the solution to 80°C for 30 minutes; then transfer it to an ice-water mixture and soak it for 5 minutes to obtain HAMA hydrogel.

[0010] The HAMA hydrogel in A2 includes HAMA, lithium phenyl phosphate and deionized water, and the volume ratio of HAMA, lithium phenyl phosphate and deionized water is 10:0.25:89.75.

[0011] The specific steps for preparing ABPCs-Evs in step 2 are: B1, obtaining germ tissue 3-7 days after antler shedding; B2, cut the embryonic tissue into 0.2 mm square tissue blocks; B3, wash the cut tissue blocks three times with PBS buffer; B4, the washed tissue blocks were placed in a digestion solution with a volume ratio of type I: type II: type III collagenase of 1:1:1 and digested at 37°C for 30 min; B5, the digested tissue blocks were transferred to a 10-cm culture dish for primary cell culture. When the cell density reached 80%, flow cytometry was performed to obtain separated cells. B6, the cells separated in B5 were co-incubated with antibodies CD31, CD45, and CD235a, and flow sorted to collect cells containing CD31-CD45-CD235a-; B7, digest and centrifuge the CD31-CD45-CD235a- cells collected in B6, incubate the separated cells with ABPCs marker antibody CX43 and antibody FGFR2, perform flow cytometry sorting, collect ABPCs, and culture them in DMEM complete medium containing 10% FBS buffer; B8, ABPCs cultured in B7 were cultured at 1.6×10 6 Cells were seeded in 75 cm² culture flasks and cultured for 48 hours in complete medium containing 10% EV-depleted FBS; B9, after the culture was completed, the cell viability was assessed using the CCK-8 kit, and the culture supernatant with cell viability exceeding 90% was collected; B10, the supernatant collected in B9 was centrifuged at 750g for 20 minutes, and then at 2000g for 30 minutes; the supernatant was centrifuged again at 16,000g for 70 minutes, the supernatant was removed to obtain EVsABPCs, EVsABPCs were resuspended in pre-cooled PBS, and centrifuged again at 16,000g for 70 minutes to further purify EVs; B11, resuspend the passivated EVsABPCs in 100 µL pre-cooled PBS, detect the concentration of EVsABPCs using NTA, and store at -80°C.

[0012] Another technical solution adopted by the present invention is the antler-DCB@ABPCs-EVs bone transplant material prepared by the preparation method of the antler-DCB@EVsABPCs bone transplant material.

[0013] The beneficial effects of the present invention are: The antler-DCB@EVsABPCs bone transplant material and preparation method of the present invention injects EVs-ABPC composite hydrogel into Antler-DCB, thereby providing the transplant material with a variety of living active molecules, while showing a strong ability to recruit endogenous stem cells and giving them phenotypic and molecular characteristics similar to ABPCs, so that Antler-DCB acquires a powerful ability to regulate osteogenesis, angiogenesis, neurogenesis and immune response, thereby providing a potential therapeutic intervention measure for the treatment of severe bone defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a diagram of the manufacture and characterization of multiple species of DCB in Example 6; Figure 2 is a schematic diagram of the physical and biological characteristics and functions of EVs in Example 6; Figure 3 This is a schematic diagram of constructing antler-inspired bone transplant-DCB@EVsABPCs in Example 6; Figure 4 This is a schematic diagram of Example 6 that antler-DCB@EVsABPCs can rapidly induce bone formation in vivo; Figure 5 This is a schematic diagram of Antler-DCB@EVsABPCs stimulating angiogenesis in Example 6; Figure 6 This is a schematic diagram of Example 6 Antler-DCB@EVsABPCs promoting neurogenesis; Figure 7 This is a schematic diagram of Example 6 Antler-DCB@EVsABPCs regulating the inflammatory response during osteogenesis. DETAILED DESCRIPTION

[0015] The present invention is described in detail below in conjunction with specific implementation modes and accompanying drawings.

[0016] Example 1 The present invention provides a method for preparing antler-DCB@EVsABPCs bone transplant material, which is specifically implemented according to the following steps: Step 1, preparing the Antler-DCB stent; Step 2, preparing a hydrogel containing 10% HAMA and ABPCs-Evs; Step 3: Take equal volumes of hydrogel containing 10% HAMA and ABPCs-EVs and mix them. Each ml of ABPCs-EVs contains 2.4×10 8 EVs.

[0017] Step 4: After mixing, shake at room temperature for 5 minutes and centrifuge at 4000 rpm for 5 minutes to expel bubbles in the system. Take Antler-DCB and immerse it 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.

[0018] Step 5, take out the Antler-DCB hydrogel containing 10% HAMA and expose it to 405nm ultraviolet light for 30 seconds to obtain antler-DCB@ABPCs-EVs, which are then frozen and stored at -80°C.

[0019] Example 2 Based on Example 1, step 1 is specifically as follows: S1, by removing the outer skin, obtaining the cancellous bone of the naturally fallen antler, and cutting the cancellous bone of the antler into suitable blocks as needed. In this embodiment, the cancellous bone of the antler is cut into 4×4 mm bone samples, and the bone samples are repeatedly frozen and thawed, and then treated with 40kHz ultrasound for 20 minutes; S2, the bone samples treated with ultrasound in S1 were immersed in ethanol and shaken on a shaker for 3 h; S3, soak the bone sample soaked in S2 in ether and shake it on a shaker for 1.5 hours; S4, soak the bone sample after soaking in S3 in a sodium hydroxide solution with a mass concentration of 20 g / L and shake it on a shaker for 12 hours, wash the bone sample with running water for 3 hours after soaking, treat it with 30% hydrogen peroxide for 24 hours after washing, and wash it again after treatment; the specific washing after treatment is: wash the bone sample with running water 3 times, each time for 0.5 hour, then soak the bone sample in a solution containing 1 mg / mL DNase for 48 hours, and after washing again, soak the sample in a solution containing 0.5 mg / mL RNase for 48 hours, and wash it with running water 3 times, each time for 0.5 hour.

[0020] S5, the cleaned bone sample was placed in a mixture of chloroform and methanol in a volume ratio of 3:1 for 1 hour, and then the bone sample was rinsed with distilled water and dried at 70°C for 1 day to produce Antler-DCB.

[0021] Example 3 Based on Example 2, the hydrogel containing 10% HAMA was prepared as follows: The hydrogel containing 10% HAMA includes HAMA, lithium phenyl phosphate (LAP) and deionized water, and the ratio of HAMA, lithium phenyl phosphate (LAP) and deionized water is 10:0.25:89.75.

[0022] A1 Prepare a 0.25% initiator standard solution, specifically: take 10 mL of PBS buffer, add it to a brown bottle containing 0.025 g of LAP, heat it in a 40-50°C water bath to dissolve it for 15 minutes, shake it several times during the process, and prepare a 0.25% initiator standard solution; A2 Preparation of a hydrogel containing 10% HAMA is as follows: (1) Place the required mass of HAMA in a glass bottle or beaker; (2) Add the required volume concentration of 0.25% initiator standard solution to the above container and stir to dissolve at room temperature for 0.5-1h; (3) Heat the solution to 80°C and maintain for 30 minutes; then quickly transfer to an ice-water mixture and soak for 5 minutes to obtain a hydrogel containing 10% HAMA.

[0023] Example 4 Based on Example 3, the preparation of ABPCs-Evs in step 2 is specifically as follows: B1, obtain the germ tissue 3-7 days after the hard horn of the antler falls off. After 3-7 days of falling off to ensure that the ABPCs in the tissue are in the proliferation stage, cut the skin covering the antler germ to expose the germ tissue underneath; B2, remove the germ tissue and cut it into 0.2 mm square tissue blocks; B3, wash the cut tissue blocks three times with PBS buffer to remove blood and other components; B4, place the washed tissue blocks in digestion solution (type I: type II: type III collagenase = 1:1:1) and digest at 37°C for 30 min; 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 obtain separated cells. B6, incubate the above separated cells with antibodies CD31, CD45, and CD235a, and perform flow cytometry sorting to collect CD31-CD45-CD235a- cells to remove blood and immune-related cells, and continue culturing; B7, digest the CD31-CD45-CD235a- cells collected above, centrifuge, co-incubate with ABPCs marker antibody CX43 and antibody FGFR2, and perform flow cytometry sorting to collect CX43+FGFR2+ cells, i.e. ABPCs, and culture them in DMEM complete medium containing 10% FBS buffer to extract EVsABPCs; B8, ABPCs cultured in B7 were cultured at 1.6×10 6 The cells were seeded in a 75 cm² culture flask and cultured for 48 hours in complete medium containing 10% EV-depleted FBS (to exclude the interference of exosomes in serum); B9, after the culture is completed, the cell viability is evaluated using the CCK-8 kit, and the culture supernatant with cell viability exceeding 90% is collected to ensure the quality of the extracted EVsABPCs; B10, the supernatant collected in B9 was centrifuged at 750g for 20 minutes and then at 2000g for 30 minutes; the supernatant was centrifuged again at 16,000g for 70 minutes, the supernatant was removed, and the obtained EVsABPCs were resuspended in pre-cooled PBS and centrifuged again at 16,000g for 70 minutes to further purify the EVs; B11, resuspend the passivated EVsABPCs in 100 µL pre-cooled PBS, detect the concentration of EVsABPCs using NTA, and store at -80°C.

[0024] Example 5 This embodiment provides a method for using antler-DCB@EVsABPCs, which is specifically implemented in the following steps: C1, 28 SD rats (about 300 grams, male) provided by the Experimental Animal Center of Air Force Medical University were used in the experiment; C2, first, 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 defect with a diameter of 4 mm and a depth of 4 mm in the middle of the femur; C6, iodine was used to clean the wound and dissipate heat; C7, a 4 mm diameter spherical stent 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 with 4-0 sutures; C9, to prevent infection, all rats were injected with 25,000 units of penicillin in the first three days after surgery.

[0025] Among them, the specific steps of the preparation method of the antler-DCB@EVs-ABPC bone transplant material of the present invention are as follows: Step 1, S1 is used to cut the antler cancellous bone into blocks of 4×4 mm in size, which is conducive to further decellularization and destroys immunogenic substances such as DNA by destroying the cell structure. The freezing and thawing method is adopted, and the principle is: the cell membrane is destroyed by forming ice crystals to increase the extractability of cell components. The 40kHz ultrasonic treatment is adopted, and its advantage is that the ultrasonic treatment can further destroy the cell structure and promote the release of cell components.

[0026] The role of S2 is that ethanol can help remove lipids and other organic substances from the sample, while helping to fix the tissue structure and reduce the impact of the components on the matrix. The role of S3 is that ether soaking can dissolve and remove lipid components in the cell membrane, and ether is volatile, which can effectively reduce the amount of solvent remaining in the tissue. The role of S4 is to dissolve organic components such as cell membranes and intracellular proteins, making cell residues easier to remove, and help degrade residual cellular components in the sample, while eliminating possible microbial contamination. The purpose of the second wash is to degrade residual DNA to reduce the risk of immune response, and to degrade residual RNA to reduce the risk of immune response, while removing fat and other fat-soluble components in bone tissue again.

[0027] Step 2: The standard solution containing 0.25% initiator configured in A1 can quickly cause HAMA cross-linking under ultraviolet light, changing it from liquid to gel; the liquid HAMA configured in A2 is mixed with EVsABPCs, and the liquid HAMA solution is required in the pores of Antler-DCB, so the HAMA needs to remain in liquid form. After the three materials are mixed, they are cross-linked by ultraviolet light.

[0028] The present invention also provides antler-DCB@ABPCs-EVs bone transplant material, which is composed of the following raw material components by mass 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 2.4×10^8EVs, 40.1%~44.7%.

[0029] The roles of the components in the antler-DCB@EVsABPCs bone transplant material of the present invention are as follows: Antler cancellous bone (Antler-DCB): The antler cancellous bone is decellularized to obtain a scaffold of the ultrastructure, biophysical and biochemical properties of the treated cancellous bone. Antler-DCB has the same crystal structure as the hydroxyapatite in other vertebral DCBs. The carbon content of Antler-DCB is significantly higher than that of different vertebral-DCBs, and exists in the form of CaCO3, which can enhance the osteogenesis of bone marrow mesenchymal stem cells (BMSC) more than hydroxyapatite. At the same time, the content of Mg, S, and Si in antler-DCB is 3.3 times higher than that in different vertebral-DCBs. These three elements are essential for bone regeneration. Antler-DCB has a stronger ability to promote new bone formation than other bone-DCBs, highlighting its potential as a natural transplant material for bone repair.

[0030] Methacryloyl hyaluronic acid (HAMA) with a mass concentration of 10g / L has a wide range of important functions. First of all, it has excellent biocompatibility. As a derivative of hyaluronic acid, HAMA inherits the good biocompatibility of hyaluronic acid, which means that it can be used safely in vivo without causing obvious immune response or rejection. Secondly, by modifying the methacrylic group on the hyaluronic acid molecular chain, it acquires photocuring ability. This property enables HAMA to form a stable hydrogel structure through photocuring technology, providing a solid foundation for its application in the biomedical field. HAMA hydrogel can also be used as a drug carrier. By controlling the structure and composition of the hydrogel, the slow release of the drug can be achieved, thereby improving the efficacy of the drug and reducing side effects.

[0031] Antler blastema progenitor cell exosomes (ABPCs-EVs): Antler blastema progenitor cells are a new type of stem cell for antler growth. The biological signals required for antler growth are transmitted by ABPC extracellular vesicles (EVs), including cytoplasmic proteins, lipids, RNA and other substances. ABPC-EVs showed similar morphology, epigenetic markers and size distribution to BMSC-EVs. RNA transcriptomics and proteomics analysis showed that the up-regulated genes of ABPC-EVs were mainly involved in cell cycle, cell movement, wound healing and angiogenesis; the down-regulated genes of ABPC-EVs were mainly involved in cell death and oxidative stress. Proteomics analysis showed that the up-regulated proteins of ABPC-EVs were mainly involved in osteogenesis, axon growth and blood circulation; the down-regulated proteins were mainly involved in inflammatory response and oxidative stress. ABPC-EVs showed a strong ability to recruit endogenous stem cells and endowed them with phenotypic and molecular characteristics similar to ABPC, which was beneficial to osteogenesis, angiogenesis, neurogenesis, immune regulation and bone regeneration.

[0032] Example 6 This example provides the test results of antler-DCB@EVs-ABPC bone transplant materials. The specific results are as follows: Figure 1 As shown, the bone structure within the antler provides structural cues to promote rapid bone growth. Using antler-DCB ( Figure 1 , A and B). Deer antler-DCB showed a porous structure similar to that of deer, cattle, and human spine-DCB ( Figure 1 , C), but its volume 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 than those of different spine-DCBs ( Figure 1, D to F). The large porous structure may promote bone ingrowth and facilitate the osteointegration of antler-DCB with the surrounding bone. In addition, the surface roughness and hydrophilicity of antler-DCB were significantly higher than those of different spine-DCBs ( Figure 1 , G and H). This facilitates cell adhesion and fluid infiltration, which helps bone regeneration. Mechanical properties are key elements of bone grafts that support regeneration. The maximum stress of antler DCB is within the mechanical range of cancellous bone, indicating that its mechanical suitability is suitable for bone regeneration ( Figure 1 , I). X-ray diffraction spectrum (XRD) analysis showed that antler-DCB has 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 spine-DCB. In addition, energy dispersive spectroscopy found that the carbon content of antler-DCB was significantly higher than that of different spine-DCB ( Figure 1 , L), FT-IR spectroscopy showed that carbon may be found in the form of CaCO3 ( Figure 1 , K). In addition, the contents of Mg, S, and Si in antler-DCB were more than 3.3 times higher than those in different spine-DCB ( Figure 1 , M to O). These three elements are essential for bone regeneration. DCBs were implanted into rat femoral defects to further evaluate their in vivo osteogenesis. At 12 weeks after 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). New bone formation in the local defect area of ​​the antler DCB group showed significantly higher bone mineral density (BMD), bone tissue volume / total tissue volume (BV / TV), and trabecular thickness (Tb.Th.) at 12 weeks ( Figure 1 , Q to T). These findings suggest 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 fabrication process and characterization techniques of decellularized marrow bone. (B) Naturally detached sika deer antler. (C) Scanning electron microscopy (SEM) image of the DCB surface. (D to F) Porosity (D), pore size (E), and specific surface area (SSA) (F) measurements in DCB (n=4). (G) Roughness (Ra) measurements of DCB using 3D atomic force microscopy (AFM). (H) Water contact angle (WCA) measurements in DCB (n=4). (I) Compressive stress-strain curves of DCB. (J) X-ray diffraction (XRD) patterns of DCB. (K) Fourier transform infrared (FT-IR) spectra of DCB. (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, new bone tissue) 12 weeks after surgery. (Q) 3D reconstruction and 2D micro-CT images show regenerated bone around the defect. (R to R) Quantitative analysis of microstructural parameters of regenerated bone tissue, including BMD (R), BV / TV (S), and Tb.Th (T) (n = 5). All statistical data are expressed as mean ± SD. Statistical analysis was performed using ANOVA and Bonferroni post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001.

[0033] like Figure 2 As shown in Figure 2, bioactive substances in growing antlers are inevitably lost during the decellularization process. Therefore, antler-DCBs were supplemented with EVsABPCs to enhance their bioactive cues related to bone regeneration. Their characteristics were characterized and compared with those of EVs from the most widely used BMSCs (EVsBMSCs). Figure 2 , A). EVsABPCs showed similar morphology, surface markers, and size distribution as 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 transcriptomic and proteomic 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 , G). Enrichment analysis showed that there were 282 upregulated genes in EVsABPCs, which were mainly involved in the cell cycle (e.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 down-regulated genes were mainly involved in cell death (e.g. Dynll1, Chmp2b, Hsp90aa1 ) and oxidative stress (e.g. Ptpn11, Hspa8, Arpc2 )( Figure 2 , E). In proteomic analysis, 227 upregulated proteins in EVsABPCs were mainly involved in osteogenesis (e.g. FOSL2, GLG1, ANXA2, RUNX2, THBS3), axon growth (e.g. CACNA1C, STMN1, OLIG1, DCLK1), and blood circulation processes (e.g. CD36, PDE3A, TTN). 143 downregulated proteins were related to inflammatory response (e.g. HP, ORM1, F12) and oxidative stress (e.g. PDGFRB, APOA4, PPIA, SLCLA1) ( Figure 2 , H and I). In general, EVsABPCs contain more powerful bioactive signals that are beneficial to tissue regeneration than EVsBMSCs, highlighting their therapeutic potential in bone injury repair. Secondly, the chemotactic effect of EVsABPCs on BMSCs migration was verified ( Figure 2 , J). In the scratch test, EVsABPCs increased the wound size recovery rate by 4.2-fold, and EVsBMSCs increased the recovery rate by 1.8-fold compared with PBS ( Figure 2 , K and M). In the transwell migration assay, the number of migrating cells observed in the EVsABPCs group was 3.5 times that of the vehicle group and 1.9 times that of the EVsBMSCs group ( Figure 2 , L, and N). These findings suggest that EVsABPCs have a stronger chemotactic effect on BMSCs than EVsBMSCs. Further transcriptome analysis showed that 416 DEGs were expressed between BMSCs treated with EVsABPCs and EVsBMSCs ( Figure 2 , O to Q). These genes are not only involved in cell motility, but also in cell cycle, bone mineralization, neurogenesis, and stem cell proliferation. These findings suggest that EVsABPCs can induce BMSCs to transform into a phenotype that is conducive to tissue regeneration. Figure 2Figure 2. (A) Schematic diagram of EVs characterization techniques. (B to D) EVs were characterized by (B) transmission electron microscopy (TEM), (C) nanoparticle tracking analysis (NTA), and (D) western blotting. (E) Heat map showing the distribution of differentially expressed genes (DEGs) between EVsABPCs and EVsBMSCs, and the enriched 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 significance. (G) Volcano plot of differentially expressed proteins (DEPs) in EVsABPCs and EVsBMSCs. (H and I) Bar graphs showing the enriched gene ontology (GO) terms of upregulated genes (H) and downregulated genes (I) in EVsABPCs and EVsBMSCs. (J) Workflow for evaluating the effects of EVs on BMSCs. (K and M) Wound healing rate of BMSCs after each treatment within 24 h (n = 4). (L and N) Analysis of BMSC migration behavior using transwell assay (n = 4). (O) Multiple volcano plots showing DEGs of BMSCs treated with PBS, EVsABPCs, and EVsBMSCs. (P) Principal component analysis illustrating the differences in BMSCs treated with PBS, EVsBMSCs, and EVsABPCs. (Q) Network diagram showing the enriched GO terms and pathways of upregulated genes in the EVsABPCs group versus the EVsBMSCs group. All statistics are presented as mean ± SD. Analyses were performed using ANOVA and Bonferroni post hoc test. ***P < 0.001.

[0034] like Figure 3 As shown in the figure, 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 hyaluronic acid methacryloyl (HAMA) hydrogels, which can be combined with versican (VCAN), a key surface marker of EVs, to achieve their gradual release ( Figure 3 , A). EVs are evenly distributed in the hydrogel, as indicated by PKH26 labeling ( Figure 3 , B). Secondly, a series of computational techniques were used to evaluate the detailed pattern of interaction between HAMA and VCAN. The results showed that VCAN / HAMA has good binding stability ( Figure 3 , C and D). In addition, analysis of the radius of gyration (Rg) showed that the binding of HAMA to VCAN had little effect on the changes in its compactness and rigidity 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 , G). The hydrogen bond (H-bond) distribution between VCAN and HAMA further confirmed the stable binding of HAMA to the EV surface ( Figure 3 , H). In addition, the negatively charged HAMA hydrogel formed a strong interfacial electronic interaction with the positively charged antler-DCB ( Figure 3 , I and J), and endowed antler-DCB with a dual-phase sponge structure and excellent pore size distribution ( Figure 3 , K and L). Notably, in situ fluorescence signals of the hydrogels containing cyanine 5.5 monoacid were observed at different times after in vivo implantation. Under in vivo conditions, the HAMA hydrogels in antler-DCB@EVsABPCs showed slower degradation kinetics than the HAMA hydrogels alone ( Figure 3 , M and N), which may be due to the extensive amide bond formation between HAMA and the minerals in antler-DCB. In addition, EVs were continuously released from antler-DCB@EVsABPCs composites for at least 18 days ( Figure 3 , O). In addition, antler-DCB@EVsABPCs exhibited excellent hydrophilicity ( Figure 3 , P) and good biocompatibility ( Figure 3 , Q), making it a potential candidate for bone repair applications. Figure 3(A) Schematic diagram of the interaction between HAMA hydrogel and EVs. (B) EVs labeled with PKH26 (red) in HAMA hydrogel were examined by z-stack scanning using confocal laser scanning microscopy. Fluorescence intensity results (left) are shown as 3D views of surface remodeling, presented in angled views (middle) and frontal views (right). (C to 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) Compound docking simulation of VCAN and HAMA hydrogels. (I) Schematic diagram of the fabrication and characterization of antler-DCB@EVs. (J) Surface potential of antler-DCB and HAMA hydrogels was quantitatively analyzed by Kelvin probe force microscopy. (K) Pore size distribution of ABPCs of antler-DCB@EVs. (L) Characterization of antler-DCB@EVsABPCs by TEM. (M and N) Degradation rates of antler-DCB@EVsABPCs and HAMA hydrogels (n=3). (O) Cumulative release of EVs was measured using bicinchoninic acid assay at days 1, 3, 6, 9, 12, 15, and 18 (n=3). (P) WCA measurement of antler-DCB@EVsABPCs. (Q) CCK-8 analysis of BMSCs cultured on different scaffolds for 1, 3, and 7 days (n=3). All statistics are presented as mean ± SD. Statistical analysis was performed using ANOVA and Bonferroni post hoc test. *P<0.05, **P<0.01, and ***P<0.001.

[0035] like Figure 4 As shown in Figure 2, a rat femoral condyle defect model was established to evaluate the osteogenic activity of antler-DCB@EVsABPCs in vivo ( Figure 4 , A). At the end of eight weeks, the bones in the untreated group had grown little and were deformed due to a lack of effective structural support ( Figure 4 , C), indicating that the size of the femoral defect exceeded the critical size for spontaneous healing. In rats implanted with antler-DCB, more new bone was observed than in untreated rats, which may be attributed to the biochemical and biophysical cues in antler-DCB. Notably, the introduction of 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 the new bone in the antler DCB@EVsABPCs group were significantly higher than those in the other groups ( Figure 4, D). In conclusion, 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 had more new bone formation, and the antler-DCB@EVsABPCs group had the most new bone tissue ( Figure 4 , B). Sequential fluorescence labeling method was used to quantify the new bone mineralization rate. The mineral apposition rate (MAR) of the antler-DCB@EVsABPCs group increased by 1.6 times compared with the antler-DCB@EVsBMSCs group and increased by 2.3 times compared with the antler-DCB group ( Figure 4 , F). In addition, 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 ( Figure 4 , G), and the lowest in the untreated group. The results confirmed that antler-DCB@EVsABPCs promoted bone formation in vivo at the histological and molecular levels. Interestingly, at 2 weeks after surgery, the number of BMSCs (CD45–DC44+CD29+) detected in the antler-DCB@EVsABPCs group was significantly higher than that in the other groups ( Figure 4 , H, and I). These findings suggest that antler-DCB@EVsABPCs recruit more BMSCs to the site of injury. The regenerated tissues of each group were collected 2wp after infection and subjected to RNAseq analysis. A total of 1,405 and 879 DEGs were observed in the regenerated tissues of the antler-DCB@EVsBMSCs and antler-DCB groups, respectively (Log2|FC|>1, P<0.5). Subsequently, cluster analysis of the combined DEGs among the three groups and GO analysis were performed, and 5 clusters with different key features and biological significance were identified ( Figure 4, J). Cluster 1 contained genes specifically enriched in the antler-DCB@EVsABPCs group, which were involved in ossification, angiogenesis, and neurogenesis. Cluster 5 contained genes specifically downregulated in the antler-DCB@EVsABPCs and antler-DCB@EVsBMSCs, which were mainly related to regulating immune and inflammatory responses. The transcriptome profiles of the regenerating tissues of the antler-DCB@EVsABPCs group were compared with those of the antler growth center, and a total of 430 shared upregulated genes were identified, which were enriched in pathways critical for rapid antler development, including genes related to the mitogen-activated protein kinase (Erk1 / 2) signaling pathway, the bone morphogenetic protein (BMP) signaling pathway, the transforming growth factor-β (TGF-β) signaling pathway, and the WNT signaling pathway. Notably, marker genes of ABPCs (e.g., Prrx1, Tnc, Dlx5, Ptn, and Sox4) were also found in the antler-DCB@EVsABPCs group, indicating that antler-DCB@EVsABPCs triggered a bone growth process similar to antler growth ( Figure 4 , K). Taken together, these results suggest that antler-DCB@EVsABPCs trigger transcriptome profiles at local injury sites, partially mimicking the molecular dynamics of antler growth centers. Figure 4 (A) Schematic diagram of exploring the osteogenic capacity of antler-DCB@EVsABPCs. (B) H&E and Masson staining images showing new bone formation 8 weeks after surgery (M, material; NB, new bone tissue). (C) 2D micro-CT (upper) and 3D reconstruction (lower) 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 summary quantitative data showing newly formed trabeculae labeled with calcein AM (green fluorescence) and alizarin red (red fluorescence) at 2 and 4 weeks after 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 was used to detect the recruitment of BMSCs on various scaffolds in vivo (n=5). (J) Cluster analysis and GO analysis results of DEGs in different groups identified key different features and biological significance. (K) Schematic diagram of the common mechanism of antler growth and antler-DCB@EVsABPCs-induced bone growth. All statistics are presented as mean ± SD. Statistical analysis was performed using ANOVA with Bonferroni post hoc test. **P < 0.01 and ***P < 0.001.

[0036] like Figure 5As shown in Figure 2, the rapid growth of antlers is accompanied by the rapid expansion of blood vessels. Consistent with this, angiogenesis-related proteins were significantly enriched in EVsABPCs ( Figure 5 , A to C). Therefore, we evaluated the effect of antler-DCB@EVsABPCs on angiogenesis in regenerating 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 effects on target cells. After 24 h of treatment with antler-DCB@EVsABPCs, the migration ability of HUVECs was significantly improved, and the number of transmembrane cells was significantly higher than that of the antler-DCB@EVsBMSCs treatment group ( Figure 5 , F, and H). In addition, more mature tubular structures and higher density of cell junctions were observed in the antler-DCB@EVsABPCs group compared with the antler-DCB@EVsBMSCs group ( Figure 5 , G and I). These findings suggest that antler-DCB@EVsABPCs can promote the migration and tube formation ability of HUVECs more than antler-DCB@EVsBMSCs. We further evaluated the effect of EVsABPCs on angiogenesis in vivo ( Figure 5 , J). Eight weeks after inoculation, the vascular structure of the antler-DCB@EVsABPCs group was most significantly improved, followed by the antler-DCB@EVsBMSCs group and the antler-DCB group. The untreated group showed the least significant improvement ( Figure 5 , K). Eight weeks after inoculation, the blood vessel 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 , L). In addition, the expression of two representative vascular markers, platelet endothelial cell adhesion molecule-1 (CD31) and endomucin (EMCN), was significantly higher in the antler-DCB@EVsABPCs group than in the other groups ( Figure 5 , M to O). Collectively, these results suggest that antler-DCB@EVsABPCs exhibit potent pro-angiogenic capacity, promoting new blood vessel formation to support extensive new bone formation. Figure 5(A to C) Protein sequencing analysis of DEPs associated with regulating angiogenesis in EVsABPCs and EVsBMSCs. (D and E) Cellular uptake assay showing that PKH26-labeled EVs were internalized and distributed in HUVECs. HUVECs were stained with phalloidin (green) and EVs were stained with PKH26 (red). (F and H) Migration behavior of HUVECs was analyzed by transwell (n = 3). (G and I) Tube-forming ability of HUVECs under each treatment (n = 3). (J) Schematic diagram of exploring the angiogenic ability of antler-DCB@EVsABPCs. (K and L) 3D reconstructed images and summarized quantitative data showing newly formed blood vessels at 4 and 8 weeks after implantation in different groups (n = 5). (M) Representative immunofluorescence images of CD31 (green), EMCN (green), and nucleus (blue) staining at eight weeks. (N and O) Quantitative analysis of all positively stained areas. All statistical data are expressed as mean ± SD. Statistical analysis was performed using ANOVA and Bonferroni's post hoc method. **P<0.01, ***P<0.001.

[0037] like Figure 6 As shown in Figure 2, neurogenic proteins such as ATP2B2, IGFBP5, GIT1, and SMG9 were significantly enriched in EVsABPCs ( Figure 6 , A to C). Therefore, the potential effects of EVsABPCs on neuronal regenerative capacity were investigated. PKH26-labeled EVs colocalized in the cell bodies and axons of neurons ( Figure 6 , D and E), indicating that EVsABPCs and EVsBMSCs can be directly internalized by neurons. In the EVsABPCs-treated group, the axon length of neurons (428.5 ± 22.7 μm) was significantly higher than that of the EVsBMSCs (363.0 ± 42.9 μm) and vehicle (258.3 ± 18.3 μm) groups ( Figure 6 , F, G, and I). A similar trend was observed in dorsal root ganglion (DRG) explants ( Figure 6 , H). 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 ( Figure 6 , J). These findings suggest that EVsABPCs have a strong ability to promote axonal growth. Subsequently, sensory nerve fibers were visualized by labeling with calcitonin gene-related peptide (CGRP), a marker for sensory nerves within new bone. Antler-DCB@EVsABPCs showed the highest CGRP expression compared to the rest of the groups ( Figure 6, K to M). This finding suggests that antler-DCB@EVsABPCs achieve greater sensory innervation, which may indirectly contribute to its beneficial effects on bone growth. The transformation of nerve fibers from sympathetic nerves to cholinergic nerves is beneficial to the survival of osteoblasts. It was found that the conversion of sympathetic nerves to cholinergic nerves in the antler-DCB@EVsABPCs group was significantly higher than that in other groups, as manifested by increased expression of the cholinergic marker vesicular acetylcholine transporter (VAChT) and decreased expression of the noradrenergic marker tyrosine hydroxylase (TH) ( Figure 6 , N to P). In addition, the acetylcholine content was the highest in the antler-DCB@EVsABPCs group, followed by the antler-DCB@EVsBMSCs group and the antler-DCB group, and was the lowest in the untreated group ( Figure 6 , Q). The beneficial effects of antler-DCB@EVsABPCs in sympathetic-cholinergic switching during new bone formation were confirmed. Figure 6 (A to C) Protein sequencing analysis of DEPs associated with neurogenesis regulation in EVsABPCs and EVsBMSCs. (D and E) Cell uptake assays showed that PKH26-labeled EVs were internalized and distributed in neurons. Neurons were stained with Tuj1 (green), nuclei were stained with DAPI (blue), and EVs were stained with PKH26 (red). (F to H) DRG neurons and explants were co-cultured with different groups of EVs for 72 h. (I) Quantification of axon 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 promoted neurogenesis and the conversion of neuronal fibers from sympathetic to cholinergic nerves. (L and M) Immunofluorescence staining showed the distribution of CGRP+ nerves in the bone regeneration area after different treatments (n = 5). (N) Representative immunofluorescence images of the cholinergic marker vesicular acetylcholine transporter (VAChT) (green), the sympathetic nerve marker tyrosine hydroxylase (TH) (green), and the nucleus (blue) stained at the bone formation site in rats. (O and P) Quantitative analysis of all positively stained areas of cholinergic and sympathetic nerves (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.

[0038] like Figure 7 As shown in Figure 2, many proteins related to inflammatory response (such as ORM1, CTSC, COL6A1, and FABP4) were downregulated in EVsABPCs compared with EVsBMSCs ( Figure 7, A to C). Therefore, the effect of EVsABPCs on the phenotypic transition of macrophages was investigated. Compared with those induced by EVsBMSCs and PBS, EVsABPCs treatment resulted in a significant decrease in the expression of iNOS (M1 marker) and a significant increase in the expression of Arg1 (M2 marker) in macrophages ( Figure 7 , D and E). In addition, EVsABPCs increased the anti-inflammatory IL-10 level and decreased the levels of pro-inflammatory factors TNF-α, PGE2, and IL-1β in BMSCs ( Figure 7 , F). The results showed that EVsABPCs were able to induce M1 to M2 macrophage polarization and alleviate inflammatory responses, which may partly contribute to their powerful beneficial effects on bone regeneration. Next, the in vivo immunomodulatory capacity of antler-DCB@EVsABPCs was evaluated within 7 days after implantation ( Figure 7 , G). The area of ​​iNOS+ cells (M1 macrophages) in the antler-DCB@EVsABPCs group was the lowest, followed by the antler-DCB@EVsBMSCs and antler-DCB groups, and the untreated group was the lowest. The area of ​​CD206+ cells (M2 phenotype) showed an opposite trend, with the antler-DCB@EVsABPCs group having the largest area ( Figure 7 , H and I). These findings confirmed that antler-DCB@EVsABPCs were able to convert M1 macrophages to M2 phenotype in vivo. In addition, compared with antler-DCB@EVsBMSCs treatment, antler-DCB@EVsABPCs treatment significantly increased the expression of the anti-inflammatory factor Arg1 and reduced the levels of pro-inflammatory factors ( Figure 7 , J and K), which are essential for bone growth. Figure 7 Figure 2. (A to C) Sequencing analysis of DEP proteins associated with inflammatory regulation in EVsABPCs and EVsBMSCs. (D) Quantification of iNOS+F4 / 80+ cell rate. (E) Quantification of Arg1+F4 / 80+ cell rate. (F) Evaluation of pro-inflammatory markers TNF-α, PGE2, IL-10, and IL-1β in macrophages by enzyme-linked immunosorbent assay (ELISA). (G) Schematic diagram of the evaluation of in vivo immune regulation of antler-DCB@EVsABPCs within 7 days after implantation. (H) Representative immunofluorescence images of iNOS (red), CD206 (green), and cell nuclei (blue) staining. (I) Quantification of all positively stained areas (n = 4). (J and K) Western blot analysis and summary quantitative data of TNF-α, COX2, and Arg1. All statistical data are presented 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 transplant material, characterized in that: Follow the steps below to implement it: Step 1, preparing the Antler-DCB stent; Step 2, preparing 10% HAMA hydrogel and ABPCs-Evs; Step 3, taking equal volumes of hydrogel containing 10% HAMA and mixing them with ABPCs-EVs; Step 4, take Antler-DCB and immerse it in 10% HAMA hydrogel containing ABPCs-EVs for 30 minutes; Step 5, take out the Antler-DCB containing 10% HAMA hydrogel and expose it to 405nm ultraviolet light for 30 seconds to obtain antler-DCB@ABPCs-EVs bone transplant material, and store it in a frozen state at -80°C.

2. The method for preparing the antler-DCB@EVsABPCs bone transplant material according to claim 1, characterized in that: The specific steps of step 1 are: S1, antler cancellous bone was cut into 4 × 4 mm bone samples and then treated with ultrasound after repeated freezing and thawing; S2, the bone samples treated with ultrasound in S1 were immersed in ethanol and shaken on a shaker for 3 h; S3, soak the bone sample soaked in S2 in ether and shake it on a shaker for 1.5 hours; S4, soaking the bone sample soaked in S3 in a sodium hydroxide solution with a mass concentration of 20 g / L and shaking on a shaker for 12 hours, washing the bone sample with running water for 3 hours after soaking, treating it with 30% hydrogen peroxide for 24 hours after washing, and washing it again after treatment; S5, the cleaned bone sample was placed in a mixture of chloroform and methanol in a volume ratio of 3:1 for 1 hour, and then the bone sample was 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 transplant material according to claim 2, characterized in that: The specific ultrasonic treatment in S1 is: using 40kHz ultrasonic waves to treat the bone sample for 20 minutes.

4. The method for preparing the antler-DCB@EVsABPCs bone transplant material according to claim 2, characterized in that: The re-cleaning in S4 is as follows: the bone sample is washed with running water for 3 times, each time for 0.5 hours, and then the bone sample is immersed in a solution containing 1 mg / mL DNase for 48 hours. After washing again, the sample is immersed in a solution containing 0.5 mg / mL RNase for 48 hours, and washed with running water for 3 times, each time for 0.5 hours.

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

6. The method for preparing the antler-DCB@EVsABPCs bone transplant material according to claim 5, characterized in that: The HAMA hydrogel in A2 comprises HAMA, lithium phenyl phosphate and deionized water, and 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 transplant material according to claim 1, characterized in that: The specific steps for preparing ABPCs-Evs in step 2 are: B1, obtaining germ tissue 3-7 days after antler shedding; B2, cut the embryonic tissue into 0.2 mm square tissue blocks; B3, wash the cut tissue blocks three times with PBS buffer; B4, the washed tissue blocks were placed in a digestion solution with a volume ratio of type I: type II: type III collagenase of 1:1:1 and digested at 37°C for 30 min; B5, the digested tissue blocks were transferred to a 10-cm culture dish for primary cell culture. When the cell density reached 80%, flow cytometry was performed to obtain separated cells. B6, the cells separated in B5 were co-incubated with antibodies CD31, CD45, and CD235a, and flow sorted to collect cells containing CD31-CD45-CD235a-; B7, digest and centrifuge the CD31-CD45-CD235a- cells collected in B6, incubate the separated cells with ABPCs marker antibody CX43 and antibody FGFR2, perform flow cytometry sorting, collect ABPCs, and culture them in DMEM complete medium containing 10% FBS buffer; B8, ABPCs cultured in B7 were cultured at 1.6×10 6 Cells were seeded in 75 cm² culture flasks and cultured for 48 hours in complete medium containing 10% EV-depleted FBS; B9, after the culture was completed, the cell viability was assessed using the CCK-8 kit, and the culture supernatant with cell viability exceeding 90% was collected; B10, the supernatant collected in B9 was centrifuged at 750g for 20 minutes, and then at 2000g for 30 minutes; the supernatant was centrifuged again at 16,000g for 70 minutes, the supernatant was removed to obtain EVsABPCs, EVsABPCs were resuspended in pre-cooled PBS, and centrifuged again at 16,000g for 70 minutes to further purify EVs; B11, resuspend the passivated EVsABPCs in 100 µL pre-cooled PBS, detect the concentration of EVsABPCs using NTA, and store at -80°C.

8. Deer antler-DCB@ABPCs-EVs bone transplant material prepared by the preparation method of deer antler-DCB@EVsABPCs bone transplant material as described in any one of claims 1-7.

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