Exosome-modified metal implant having immunomodulatory and osteoinductive functions, and preparation method therefor and use thereof

Abstract

The present invention belongs to the field of new biomedical materials, and specifically relates to an exosome-modified metal implant having immunomodulatory and osteoinductive functions, and a preparation method therefor and the use thereof. In the present invention, inspired by marine mussels, a mussel-derived adhesion polypeptide having a catechol group is designed by means of combining modification strategies of metal-phenol coordination chemistry with bioorthogonal click chemistry for a mussel-like adhesion molecule. An implant is modified by means of catechol / titanium coordination interaction, and linked to an azide-modified exosome so as to non-destructively modify the exosome on the surface of a medical metal implant material, resulting in an exosome-modified metal bone implant material; and the surface osteointegration efficiency of the implant material is improved by means of the immunomodulatory function and osteoinductive activity provided by the implant. A new technical method is provided for efficient modification of functionalized extracellular vesicles on the surface of a metal implant, which method has a profound effect especially on improving the survival rate of prosthetic implants in diabetic patients, and has broad prospects in the medical field.

Description

An exosome-modified metal implant with immune regulation and bone induction functions, and its preparation method and application Technical Field

[0001] The present invention belongs to the field of new biomedical materials, and specifically relates to an exosome-modified metal implant with immune regulation and bone induction functions, and a preparation method and application thereof. Background Art

[0002] Extracellular vesicles (e.g., exosomes) are vesicles containing a phospholipid bilayer structure that can be secreted by almost all cell types. These vesicles can participate in pathological processes such as inflammation, autoimmune response, endothelial dysfunction / injury, procoagulant, angiogenesis, intimal hyperplasia, and osteoblastic differentiation, and play a vital role in cell therapy. Exosomes often carry a variety of functional groups such as cytokines, growth factors, signaling lipids, DNA, and regulatory miRNAs. Among them, exosomes derived from mesenchymal stem cells (MSCs) in early osteoblastic differentiation (MSC-EVs) have shown positive effects in anti-inflammatory, cell adhesion, and osteoblastic differentiation, making them ideal regulators of bone integration. Compared with other stem cells, protein drugs, and synthetic chemicals, the natural multicomponents of MSC-EVs can induce complex and long-lasting cellular responses in a safe, low-cost, and efficient manner without being limited by immune and biological toxicity, which has obvious advantages for improving the surface bone integration of prostheses.

[0003] Prosthetic implants are one of the most successful therapeutic strategies for restoring bone and joint function. However, the immune response to prosthetic implants can hinder bone integration, compromising the effectiveness of osseointegration and leading to increased orthopedic implant failure rates. During prosthetic implant treatment, bone biological quality is maintained through bone remodeling. Bone metabolism and immune inflammatory responses are the primary factors influencing osteogenesis during this remodeling process. Bone metabolism is characterized by decreased bone formation and reduced circulating levels of biochemical markers of bone formation. Besides bone metabolism, a more significant factor influencing osteogenesis is the inflammatory immune response. Studies have shown that inflammatory cytokines released by immune cells can be detected in patients with prosthetic implants, with macrophages playing a key role. After implantation, exogenous biomaterials exacerbate the local immune response. Within hours of surgery, macrophages are recruited to the implant surface and trigger an early inflammatory cascade. Prolonged inflammatory exposure can lead to the formation of a surrounding annulus fibrosus, a key factor affecting prosthetic survival and creating a barrier to osseointegration. Furthermore, prolonged exposure to this inflammatory environment can impair MSC activity and osteogenic differentiation. At present, the integration between the prosthesis and the bone and bone remodeling are still severe challenges that affect clinical efficacy. There is an urgent need to develop effective strategies to promote bone remodeling around the prosthesis in patients.

[0004] Existing studies have shown that promoting bone formation through immune regulation can improve bone integration on the prosthetic surface. However, there is still room for exploration of flexible modification strategies based on cells, vesicles or macromolecular bioactive substances. In particular, the process of obtaining high-performance prostheses by modifying medical implants with biomass is seriously insufficient. It is urgent to develop novel and effective strategies for using MSC-EVs to modify the implant surface to improve bone integration on the prosthetic surface.

[0005] Summary of the Invention

[0006] In order to solve some problems in the prior art, the present invention provides an exosome-modified metal implant with immune regulation and osteoinduction functions, as well as its preparation method and application. The present invention synthesizes a mussel-derived polypeptide (DOPA) containing a bio-clickable group DBCO that can adhere to the surface of titanium-based materials and undergo a click reaction with the azide group linked to the exosomes. x ‐PEG5‐DBCO); The present invention synthesizes a peptide that is linked to a clickable, highly active phospholipid poly (ethylene glycol) derivative 1,2-distearoyl-sn-glycero-3-hydroethanolamine phosphate azide (DSPE-PEG) that is pre-differentiated MSC-exosomes. 2k -Azido) to modify titanium-based medical implants; the exosome-modified medical implant with dual functions of immune regulation and bone induction of the present invention can regulate macrophage polarization in vivo and in vitro, and promote peri-prosthetic bone integration.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides an exosome-modified medical implant with dual functions of immune regulation and bone induction. The exosome-modified medical implant is modified on the surface of a titanium-based material with mussel-derived adhesion polypeptides and click-chemistry modification of the azidated exosome surface to achieve the dual functions of immune regulation and bone induction.

[0009] Preferably, the exosome-modified medical implant is modified on the titanium-based material surface with a mussel-derived polypeptide (DOPA) that can click-react with the azide group linked to the exosomes. x ‐PEG5‐DBCO; exosomal DSPE-PEG after azidation 2k -Azido and (DOPA) x ‐PEG5‐DBCO achieves stable and lossless modification of exosomes through cycloaddition bioorthogonal click chemistry reaction.

[0010] In a specific embodiment, the present invention further provides a method for preparing an exosome-modified medical implant having dual functions of immune regulation and bone induction, the method comprising the following steps:

[0011] (1) Design and preparation of mussel-derived adhesion peptides: A solid-phase peptide synthesis method was used to synthesize mussel-derived peptides from the copper-free click chemistry reagent dibenzocyclooctyne (DBCO), acetone, and Fmoc-protected levodopa amino acid Fmoc-DOPA (acetonide)-OH. The resulting mussel-derived peptide was designated as (DOPA) x ‐PEG5‐DBCO;

[0012] (2) Derivatization of the surface of medical implants with peptides: The medical implants were immersed in the (DOPA) obtained in step (1) using a solution immersion method. x -PEG5-DBCO solution, and the DOPA groups are fully adhered to the implant surface to obtain a DOPA-implant;

[0013] (3) Azide modification of exosome surface:

[0014] MSC-derived exosomes (EVs) were conjugated with the azide-coupled reagent 1,2-distearoyl-sn-glycero-3-hydroethanolamine phosphate (DSPE-PEG 2k -Azido) mixture, exosomes EV combined with DSPE to obtain azidated exosomes, recorded as EV-DSPE-PEG 2k -Azido;

[0015] (4) Click chemistry modification of exosomes on the surface of medical implants: The DOPA-implant obtained in step (2) was immersed in the EV-DSPE-PEG obtained in step (3). 2k In the -Azido solution, the azide group and the DBCO group reacted via a cycloaddition bioorthogonal click chemistry reaction to achieve specific loading of exosomes, thereby obtaining EV-DOPA-modified exosome-modified medical implants with dual functions of immune regulation and bone induction.

[0016] In step (1), the structural formula of the mussel-derived polypeptide sequence is: Ac-[(DOPA)-G] x / 2 -K[(PEG5)‐(Mpa)‐(Mal‐DBCO)]‐[(DOPA)‐G] x / 2 , wherein x is the number of repeating units of DOPA amino acid, and x can be selected from 2, 4, 6 or 8.

[0017] In the specific structure, the mussel-derived polypeptide synthesized in step (1) contains spaced glycine (G) or lysine (K), and the levodopa amino acid (DOPA) is separated by a single glycine or lysine; and with the help of the side chain amino group of lysine (K), the clickable diphenylcyclooctyne group is connected through the bridging effect of short-chain polyethylene glycol (PEG5).

[0018] During the synthesis of the mussel-derived adhesion polypeptide in step (1), the dopa groups within a single molecule of the polypeptide are separated by a single glycine; the overall chain length of the mussel-derived polypeptide is 5, 9, 13 or 17 amino acid groups, and the connection mode is linear.

[0019] The present invention uses acetone as a protecting agent to protect the catechol group in levodopa amino acid (DOPA).

[0020] The medical implant described in step (2) can be made of titanium alloy material, preferably titanium sheet, titanium rod or titanium nail. x The concentration of the PEG5-DBCO solution is 0.005 mg / ml-1.000 mg / ml, and the sufficient soaking time is 12-36 hours.

[0021] The EV and DSPE-PEG in step (3) 2k -The quantity ratio of Azido is 1:100 to 1:10000.

[0022] EV-DSPE-PEG described in step (4) 2k The concentration of the Azido solution is 0.01-1.0 mg / mL, and the soaking time is 6-24 hours.

[0023] Furthermore, the present invention also provides the use of the exosome-modified medical implant having the dual functions of immune regulation and bone induction as described above in the field of preparing artificial prosthetic implant materials.

[0024] Preferably, the exosome-modified medical implant is used in the field of preparing artificial prosthetic implant materials for diabetic patients.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] Inspired by marine mussels, this invention combines the metal-phenol coordination chemistry of mussel-like adhesion molecules with a bioorthogonal click chemistry modification strategy to design a mussel-derived adhesion polypeptide with catechol groups. Metal Ti implants are modified through a powerful catechol / titanium (Ti) coordination interaction and then linked to azide-modified exosomes to create exosome-modified medical implants with both immunomodulatory and osteoinductive properties. The exosomes are non-destructively modified onto the surface of medical metal implants, imparting both immunomodulatory and osteoinductive activity to the implants.

[0027] This invention modifies the surface of bone implants with exosomes derived from mesenchymal stem cells (MSCs) that possess multiple bioactivities and are undergoing early osteogenic differentiation, through a mussel-mimicking adhesion mechanism and a bioorthogonal click reaction. This modulates the polarized phenotype of macrophages, modulating the immune-osteogenic cascade and thereby improving implant performance. It can alleviate inflammatory responses in hyperglycemic microenvironments, reducing the expression of inflammatory factors such as IL-1β, IL-6, and TNF-α; promote osteogenic differentiation of BMSCs in hyperglycemic microenvironments; and enhance periprosthetic bone integration, bone formation, and bone density in diabetic patients. The resulting exosome-modified medical implants exhibit excellent biocompatibility and promote macrophage polarization toward the anti-inflammatory M2 phenotype, synergistically improving immune microenvironmental induction at the bone-implant interface and optimizing osteogenic performance and osseointegration. Compared to the complexity, potential toxicity, and destructiveness of traditional chemical modification, the mussel-mimicking adhesion and click modification strategy described in this invention offers a rapid, mild, and structurally stable preparation method for exosomes. This method provides a new approach for efficiently modifying the surfaces of medical metal implants with functionalized exosomes. This approach has a profound impact on improving the survival rate of diabetic patients undergoing implanted prostheses, and holds broad promise in the medical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG1 is a flow cytometry analysis of primary MSC markers;

[0029] Figure 2 is an exosome identification diagram; in the figure, A is the NTA particle size analysis and transmission electron microscopy TEM image; B is the exosome marker protein expression identification diagram;

[0030] Figure 3 is a schematic diagram of the molecular structure of the components of mussel-derived peptides; in the figure, a is a representative mussel-derived peptide (DOPA)6-PEG5-DBCO molecular formula, b is DSPE-PEG 2k -Azido molecular formula, c is the cycloaddition reaction formula;

[0031] FIG4 is a representative HPLC analysis and MS analysis of (DOPA)6-PEG5-DBCO;

[0032] Figure 5 is DSPE-PEG 2k -NMR analysis chart of Azido;

[0033] Figure 6 is a FITC fluorescence microscope direct observation image;

[0034] FIG7 is an atomic force microscope (AFM) surface morphology detection diagram;

[0035] FIG8 is an X-ray photoelectron spectroscopy (XPS) surface element composition detection diagram;

[0036] FIG9 is a water contact angle test (WCA) diagram;

[0037] Figure 10 is a scanning electron microscope (SEM) image of MSC and RAW264.7 cells;

[0038] FIG11 is a graph showing average cell spreading area analysis and cell proliferation activity detection;

[0039] Figure 12 is a live-dead staining image of cells (**p<0.01);

[0040] FIG13 is a fluorescence image of reactive oxygen species detected by the direct osteogenesis-promoting effect of EV-DOPA;

[0041] FIG14 is a flow cytometry diagram showing the direct osteogenesis-promoting effect of EV-DOPA on reactive oxygen species;

[0042] FIG15 is a staining diagram showing the direct osteogenesis effect of EV-DOPA: A is ALP staining; B is ARS staining;

[0043] FIG16 is a graph showing the direct osteogenesis-promoting effect of EV-DOPA on ALP activity and ARS absorbance;

[0044] FIG17 is a graph showing RT-qPCR detection of genes related to osteogenic differentiation by EV-DOPA directly promoting osteogenesis;

[0045] Figure 18 is a graph showing the immunofluorescence staining results of related proteins Runx2 and OCN expression;

[0046] FIG19 is a schematic diagram of an experiment on EV-DOPA regulating macrophage polarization phenotype transformation;

[0047] Figure 20 is an image of macrophage iNOS and F4 / 80 immunofluorescence co-staining;

[0048] Figure 21 is an image of macrophage Arg-1 and F4 / 80 immunofluorescence co-staining;

[0049] Figure 22 is an analysis graph of relative mean fluorescence intensity of immunofluorescence staining;

[0050] FIG23 is a flow cytometry quantitative analysis of CD86 macrophage M1 polarization phenotype;

[0051] FIG24 is a flow cytometry quantitative analysis of CD206 macrophage M2 polarization phenotype;

[0052] Figure 25 is a graph showing RT-qPCR detection of inflammatory cytokines;

[0053] FIG26 is a diagram showing the expression analysis of inflammatory factors by high-throughput sequencing;

[0054] Figure 27 is a staining diagram under EV-DOPA regulated immune conditions: In the figure, A is ALP staining; B is ARS staining;

[0055] FIG28 is a graph showing the ALP positive staining area and ARS staining absorbance detection under EV-DOPA regulated immune conditions;

[0056] FIG29 is a graph showing RT-qPCR detection of osteogenic differentiation-related genes under EV-DOPA regulated immune conditions;

[0057] Figure 30 is an image of immunofluorescence staining of osteogenic differentiation-related proteins Runx2 and OCN;

[0058] FIG31 is a graph showing blood glucose monitoring in DM rats: A. Fasting blood glucose test at each age stage; B. Glucose tolerance test;

[0059] FIG32 is a CD86 immunofluorescence co-staining image and mean fluorescence intensity analysis diagram; FIG33 is a CD206 immunofluorescence co-staining image and mean fluorescence intensity analysis diagram;

[0060] FIG34 is a three-dimensional reconstruction of bone mass and bone volume fraction analysis of healthy rats and DM rats before and after titanium rod implantation; ...

[0061] FIG35 is a Micro-CT bone 3D reconstruction image of the effect of EV-DOPA on periprosthetic bone integration in DM rats;

[0062] FIG36 is an analysis of bone volume fraction, bone density, and trabecular thickness of EV-DOPA on periprosthetic bone integration in DM rats;

[0063] FIG37 is an H&E staining image showing the effect of EV-DOPA on periprosthetic bone integration in DM rats;

[0064] Figure 38 is a toluidine blue staining image;

[0065] FIG39 is an analysis diagram of annulus fibrosus thickness and bone-prosthesis contact ratio;

[0066] FIG40 is a calcein fluorescence image showing the effect of EV-DOPA on periprosthetic bone formation indices in DM rats;

[0067] FIG41 is an image of immunofluorescence staining of OCN and COL1A1 proteins. DETAILED DESCRIPTION

[0068] The present invention is further described by the following examples, however, the scope of the present invention is not limited to these examples. The present invention provides general and / or specific descriptions of the materials and experimental methods used in the experiments. Experimental methods in the following examples where specific conditions are not specified were generally performed under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, the reagents, biological materials, etc. used in the following examples were commercially available.

[0069] Osteogenic induction medium was purchased from Shanghai Xiaopeng Biotechnology Co., Ltd., Fmoc-DOPA (acetonide)-OH was purchased from Qiangyao Biotechnology Co., Ltd., DSPE-PEG2k-Azido was purchased from Xi'an Ruixi Biotechnology Co., Ltd., ALP alkaline phosphatase staining kit was purchased from Biyuntian Biotechnology Co., Ltd., NanoDrop-2000 was purchased from ThermoFisher, PrimeScript RT MasterMix, ExTaq TM The primary antibody working solution, secondary antibody working solution, goat serum blocking solution, I antibody and II antibody were purchased from Takara. SD rats were obtained from the Animal Experiment Center of Soochow University.

[0070] The present invention provides a method for preparing an exosome-modified medical implant having dual functions of immune regulation and bone induction, the method comprising the following steps:

[0071] (1) Design and preparation of mussel-derived adhesion peptides: A solid-phase peptide synthesis method was used to synthesize mussel-derived peptides from the copper-free click chemistry reagent dibenzocyclooctyne (DBCO), acetone, and Fmoc-protected levodopa amino acid Fmoc-DOPA (acetonide)-OH. The resulting mussel-derived peptide was designated as (DOPA) x ‐PEG5‐DBCO; the structural formula of the amino acid sequence of the levodopa amino acid is: Ac‐[(DOPA)‐G]x / 2-K[(PEG5)‐(Mpa)‐(Mal‐DBCO)]‐[(DOPA)‐G]x / 2, wherein x is the number of repeating units of DOPA amino acid, and x can be selected from 2, 4, 6 or 8; the overall chain length of the mussel-derived adhesion polypeptide is 5, 9, 13 or 17 amino acid groups, and the connection mode is linear.

[0072] (2) Derivatization of the surface of medical implants with peptides: The medical implants were immersed in the (DOPA) obtained in step (1) using a solution immersion method. x -PEG5-DBCO solution (concentration of 0.005 mg / ml-1.000 mg / ml) is immersed for 12h-36h, and the DOPA group is fully adhered to the implant surface to obtain a DOPA-implant; the medical implant can be selected from medical titanium sheets, titanium rods, titanium nails or other conventional prosthetic titanium alloy materials that can be used for implantation in the art.

[0073] (3) Azide modification of exosome surface:

[0074] MSC-derived exosomes (EVs) were conjugated with 100-10,000 times the amount of the azide coupling reagent 1,2-distearoyl-sn-glycero-3-hydroethanolamine phosphate (DSPE-PEG 2k -Azido) mixture, exosomes EV combined with DSPE to obtain azidated exosomes, recorded as EV-DSPE-PEG 2k -Azido;

[0075] (4) Click chemistry modification of exosomes on the surface of medical implants: The DOPA-implant obtained in step (2) was immersed in the EV-DSPE-PEG obtained in step (3). 2k -Azido solution (concentration of 0.01-1.0 mg / mL) for 6h-24h, the azide group and the DBCO group reacted through a cycloaddition bioorthogonal click chemistry reaction to achieve specific loading of exosomes, obtaining EV-DOPA-modified exosome-modified medical implants with dual functions of immune regulation and bone induction.

[0076] Example 1: Extraction and identification of exosomes from pre-differentiated MSCs

[0077] 6-8 week old Sprague-Dawley rats (weighing 200 g, SPF grade, Soochow University Animal Experimental Center) were purchased and anesthetized and sacrificed to extract primary bone marrow mesenchymal stem cells (BMSCs) from their long bones. After 3 days of culture in osteogenic induction medium containing exosome-free serum, the supernatant was extracted and centrifuged to remove cell debris. Subsequently, exosomes were extracted by ultracentrifugation. Exosomes were analyzed by NTA particle size analysis and protein marker detection.

[0078] Figure 1 is a flow cytometry identification diagram of primary MSC markers; Figure 2 is an exosome identification diagram; in the figure, A is the NTA particle size analysis and transmission electron microscopy TEM image; B is the exosome marker protein expression identification diagram; as can be seen from Figures 1 and 2, the flow cytometry identification results show that MSCs cells are CD34 and CD45 negative and CD29 and CD90 positive; the NTA particle size analysis results show that the diameter distribution of the extracted microvesicles is concentrated in 100-200nm; the exosome markers CD9, CD63, and TSG101 are tested positive, indicating that the exosomes of primary bone marrow mesenchymal stem cells have been successfully obtained.

[0079] Example 2: Preparation of EV-DOPA Coated Implants

[0080] (1) Synthesis of mussel-derived peptides

[0081] A mussel-derived peptide was synthesized using acetone-protected Fmoc-DOPA(acetonide)-OH by solid-phase peptide synthesis. Its structural formula is: Ac-[(DOPA)-G]x / 2 -K[(PEG5)‐(Mpa)‐(Mal‐DBCO)]‐[(DOPA)‐G] x / 2 , referred to as: (DOPA)x-PEG5-DBCO, where x is the number of repeating units of DOPA amino acid. In the specific structure, the mussel-derived polypeptide contains spaced glycine (G) or lysine (K), through which the levodopa amino acid (DOPA) is spaced apart; and with the help of the side chain amino group of lysine (K), the clickable diphenylcyclooctyne (DBCO) group is connected through the bridging effect of short-chain polyethylene glycol (PEG5). Figure 3 is a schematic diagram of the molecular structure of the components of the mussel-derived peptide; in the figure, a is a representative mussel-derived peptide (DOPA)6-PEG5-DBCO molecular formula, and b is DSPE-PEG 2k -Azido molecular formula, c is the cycloaddition reaction formula; Figure 4 is a representative HPLC analysis and Mass Spectrometry (MS) analysis of (DOPA)6-PEG5-DBCO; As shown in Figure 4, the HPLC analysis results indicate that the purity of the synthesized polypeptide is 98.35%, and the obtained polypeptide is consistent with the designed molecular weight.

[0082] (2) Peptide treatment of titanium alloy implants

[0083] Medical titanium sheets were immersed in a (DOPA)6-PEG5-DBCO solution (0.005-1.000 mg / ml) for 24 hours to allow the DOPA groups to fully adhere to the titanium surface. Ungrafted free peptides were washed away with ultrapure water to obtain a peptide-treated implant, designated DOPA-DBCO-Ti.

[0084] (3) Azide of exosomes

[0085] The exosomes derived from MSCs during osteogenic differentiation obtained in Example 1 were mixed with DSPE-PEG 2k -Azido was mixed and combined at a ratio of 1:100 to 1:10000 to obtain azide-modified exosomes, which were recorded as EV-DSPE-PEG 2k Figure 5 is an NMR analysis diagram of DSPE-PEG2k-Azido; Figure 5 shows that the NMR analysis of DSPE-PEG2k-Azido 2k -The main groups contained in Azido.

[0086] (4) Preparation of EV-DOPA-coated implants

[0087] The azidated exosome solution obtained in step (3) was added to step (2) at a ratio of 3:1 (w:w) and fully click-linked with the titanium-based material after DOPA adhesion for 12 hours. The specific loading of exosomes was achieved through a cycloaddition bioorthogonal click chemistry reaction between azide and diphenylcyclooctyne (DBCO) groups, resulting in an exosome-modified medical implant material with dual immune regulation and bone induction functions (EV-DOPA), which was designated EV-DOPA-Ti.

[0088] Example 3: Preparation of fluorescently labeled EV-DOPA coating modified implants

[0089] In order to better observe the implant modification effect, this embodiment provides an optional fluorescent labeled EV-DOPA modified coating preparation scheme. The difference between this embodiment and Example 2 is that the azidated exosome solution DSPE-PEG obtained in step (3) is added to the DSPE-PEG 2k -Azido and DSPE-PEG containing a visible fluorescent group 2k FITC was dissolved in sterile PBS at a ratio of 3:1 (w:w) to form a mixed solution. Exosomes derived from MSCs during osteogenic differentiation were then mixed with the mixed solution at a molar concentration of 1:1000. After sufficient fusion, azide-modified EVs were obtained, which were recorded as FITC / (2-Azido)-PEG. 2k ‐DSPE‐EV; FITC / (2‐Azido)‐PEG 2k ‐DSPE-EV reacted with DOPA-DBCO-Ti to obtain a labelable exosome-modified medical implant material with dual functions of immune regulation and bone induction, which was labeled as FITC-EV-DOPA-Ti.

[0090] Example 4: EV-DOPA coating modified implant surface properties

[0091] The surface properties of implants obtained by the EV-DOPA coating modification strategy and implants modified by other methods were compared and verified. The EV-DOPA coating modified implants prepared in Example 3 and other comparative implants were respectively placed under a fluorescence microscope for observation. Among them, the Control group is a titanium sheet soaked in ordinary PBS, the EV group is a titanium sheet modified only with exosomes, and the DOPA group is a titanium sheet modified only with DOPA groups obtained in step (2) of Example 1. Figure 6 is a direct observation image of FITC fluorescence microscope; as can be seen from Figure 6, green dot-like fluorescence was observed on the surface of FITC-EV-DOPA titanium carrying FITC groups, while no fluorescence was observed in other groups. Figure 7 is an atomic force microscope (AFM) surface morphology detection image; as can be seen from Figure 7, compared with the titanium sheet soaked in ordinary PBS, the surface roughness of the DOPA group increased, the EV group had no obvious changes, and a large number of exosome spherical protrusions were observed in the EV-DOPA group. Figure 8 is an X-ray photoelectron spectroscopy (XPS) surface element composition detection diagram; it can be seen from Figure 8 that compared with the titanium sheet soaked in ordinary PBS, the "N" element content on the surface of the modified titanium-based material is significantly increased; Figure 9 is a water contact angle detection (WCA) diagram; it can be seen from Figure 9 that compared with the titanium sheet soaked in ordinary PBS, the water contact angle of the EV-DOPA group is significantly reduced, the hydrophilicity is better, and EV is effectively modified on the surface of the titanium alloy without destroying the overall structure of EV.

[0092] MSCs and RAW264.7 cells (homemade) were seeded on the titanium surfaces of each group. Figure 10 is a scanning electron microscopy (SEM) image of MSCs and RAW264.7 cells; Figure 11 is an analysis of the average cell spreading area and a cell proliferation activity detection image; Figure 12 is a cell live-dead staining image (**p<0.01); As can be seen from Figures 10-12, the SEM images show that the cells in the EV-DOPA group spread more completely. No obvious dead cells were observed in the live-dead staining. The results of the cell proliferation detection showed that the cell proliferation activity in the EV-DOPA group was higher than that in the Control group. The above results show that EVs were successfully modified on the surface of the implant and had cell attachment and spreading properties, showing good biocompatibility.

[0093] Example 4: Characterization of EV-DOPA's in vitro osteogenic properties

[0094] (1) Antioxidative stress effect of EV-DOPA

[0095] 6-8 week old SD rats were purchased and anesthetized and killed to extract primary MSCs from the long bones. The cells were inoculated onto the control group (titanium sheet soaked in ordinary PBS), the EV group (titanium sheet modified only with exosomes), the DOPA group (titanium sheet modified only with DOPA groups obtained in step (2) of Example 1), and the EV-DOPA group (EV-DOPA-Ti), and osteogenic induction culture was performed in a high-sugar and inflammatory microenvironment simulating diabetic (DM) conditions. After 24 hours, the culture medium was aspirated and labeled using the Biyuntian Reactive Oxygen Detection Kit according to the instructions. The images were then collected using a fluorescence microscope and quantitatively analyzed by flow cytometry.

[0096] Figure 13 is a fluorescence image of reactive oxygen species detected by the direct osteogenesis effect of EV-DOPA; Figure 14 is a flow cytometry quantitative image of reactive oxygen species detected by the direct osteogenesis effect of EV-DOPA. It can be seen from Figures 13 and 14 that the results of reactive oxygen species fluorescence and flow cytometry detection show that under the DM microenvironment, more reactive oxygen species are produced in the Control group and the EV group, and compared with the Control group, the reactive oxygen species produced in the BMSCs of the EV-DOPA group are significantly reduced.

[0097] (2) ALP and ARS staining

[0098] Primary MSC cells were inoculated into the control group, EV group, DOPA group, and EV-DOPA group, respectively, and induced to differentiate into osteoblasts under the DM microenvironment. On the 7th day of induction, the culture medium was removed, washed with PBS, and then stained with an ALP alkaline phosphatase staining kit, and images were collected under an optical microscope. Positively stained cells appeared blue. On the 21st day of induction, the culture medium was removed, fixed, and stained with ARS alizarin red staining solution, and images were collected under an optical microscope. Figure 15 is a staining diagram of the direct osteogenic effect of EV-DOPA: In the figure, A is ALP staining; B is ARS staining; Figure 16 is an ALP activity detection diagram and ARS absorbance detection diagram of the direct osteogenic effect of EV-DOPA; As can be seen from Figures 15 and 16, positively stained calcium nodules appeared red, the ALP level in the control group was low, and compared with the control group, the oxidative stress level of MSC cells in the EV-DOPA group was significantly reduced, and the ALP positive staining area and ALP activity were significantly increased (***p<0.001). There was no obvious calcium nodule formation in the control group, while in comparison, the EV-DOPA group had significantly more calcium nodule-positive staining and higher absorbance values ​​(***p<0.001).

[0099] (3) Osteogenic differentiation indicators

[0100] RT-qPCR was used to characterize osteogenic differentiation. Total RNA was extracted using TRIzol. mRNA concentration and purity were assessed using a NanoDrop-2000. PrimeScript RT Master Mix was used for reverse transcription. SYBR® 1 premix, ExTaq TM A total of 2 μl of cDNA product was used for subsequent RT-qPCR analysis. Gene expression analysis was performed using the CFX96 Touch Real-Time PCR Detection System.

[0101] Gene and protein analysis of osteogenic differentiation-related markers showed that EV-DOPA treatment promoted the expression of osteogenic genes (Alp, Runx2, Ocn, Col1a1, Sp7), as well as the protein expression of Runx2, an early marker of osteogenic differentiation, and OCN, a mid- to late-stage marker. RAW264.7 cells were then seeded onto titanium surfaces in different groups and cultured in a simulated DM microenvironment. Figure 17 shows the RT-qPCR analysis of osteogenic differentiation-related genes directly induced by EV-DOPA; Figure 18 shows the immunofluorescence staining results for Runx2 and OCN. As shown in Figures 17 and 18, RT-qPCR results showed that the EV-DOPA-treated group had lower expression of proinflammatory cytokines compared to the control group. In the DM microenvironment, the gene expression levels of Alp, Runx2, Ocn, Col1a1, and Sp7 were significantly increased in the EV-DOPA group compared to the control group (*p < 0.05, **p < 0.01, ***p < 0.001). Protein immunofluorescence staining revealed increased expression of Runx2 in the early stage and OCN in the mid- to late stage in the EV-DOPA group compared with the control group (*p<0.05, **p<0.01, ***p<0.001). These results confirm that EV-DOPA treatment inhibits the differentiation of pro-inflammatory M1 macrophages and increases the number of anti-inflammatory M2 macrophages. EV-DOPA has direct antioxidant and osteogenic properties.

[0102] Example 5: EV-DOPA regulates macrophage polarization-mediated immune responses in vitro

[0103] (1) Changes in macrophage polarization phenotype

[0104] Macrophage polarization was characterized by immunofluorescence staining. Figure 19 is a schematic diagram of the experiment on EV-DOPA regulating the phenotypic transformation of macrophage polarization. The specific operation is as follows: remove the cell culture medium, wash with PBS, add 4% paraformaldehyde fixative, and fix on ice for 15 minutes. Then wash with PBS three times, add Triton solution for 10 minutes, and wash with PBS. Add immunostaining blocking solution, block on ice for 1 hour, add primary antibody working solution, and incubate at 4°C for 12 hours. Recover the primary antibody, wash with PBS three times, add fluorescent secondary antibody working solution, and incubate at room temperature in the dark for 1 hour. After sealing with anti-fluorescence quenching sealing agent, collect images under a fluorescence confocal microscope.

[0105] Figure 20 is an immunofluorescence co-staining image of macrophage iNOS and F4 / 80; Figure 21 is an immunofluorescence co-staining image of macrophage Arg-1 and F4 / 80; Figure 22 is an analysis of the relative mean fluorescence intensity of immunofluorescence staining; As can be seen from Figures 20-22, the results of cell immunofluorescence staining show that under the DM microenvironment, the expression of iNOS in macrophages is relatively high, while the expression of Arg-1 is relatively low; compared with the Control group, the expression of iNOS in cells in the EV-DOPA group was reduced, while the expression of Arg-1 was increased. Figure 23 is a flow cytometric analysis of the macrophage M1 polarization phenotype CD86; Figure 24 is a flow cytometric analysis of the macrophage M2 polarization phenotype CD206; as can be seen from Figures 23 and 24, compared with the Control group, the proportion of CD11b / CD86-positive M1 macrophages in the EV-DOPA group was significantly decreased, while the proportion of CD11b / CD206-positive M2 macrophages was increased (*p<0.05, **p<0.01, ***p<0.001).

[0106] (2) Inflammatory cytokines

[0107] RT-qPCR was used to test and characterize inflammatory cytokines, and the specific implementation was the same as step (3) in Example 4. Figure 25 is a RT-qPCR detection diagram of inflammatory cytokines; as shown in Figure 25, the RT-qPCR results show that under the DM microenvironment, compared with the Control group, the gene expression levels of proinflammatory cytokines IL-1β, IL-6 and TNF-α in the EV-DOPA group were significantly reduced, while the anti-inflammatory IL-10 gene expression was increased (*p<0.05, **p<0.01, ***p<0.001). Figure 26 is a high-throughput sequencing analysis of inflammatory factor expression; as shown in Figure 26, through the analysis of high-throughput sequencing results, EV-DOPA can significantly reduce the expression of most inflammatory-related factors under the DM microenvironment. The results confirmed that EV-DOPA can regulate the immune response mediated by macrophage polarization.

[0108] Example 6: EV-DOPA indirectly promotes osteoblast differentiation in vitro through immune regulation

[0109] (1) ALP and ARS staining

[0110] The ALP and ARS staining operation is the same as step (2) in Example 4. After the EV-DOPA-modified prosthesis is co-cultured with the conditioned medium prepared by macrophage culture, Figure 27 is a staining diagram under the immune conditions regulated by EV-DOPA: In the figure, A is ALP staining; B is ARS staining; Figure 28 is a detection diagram of the ALP positive staining area and ARS staining absorbance under the immune conditions regulated by EV-DOPA; As can be seen from Figures 27 and 28, CM Control The ALP level in the group was low; Control Compared with the CM group, EV-DOPA The ALP positive staining area and ALP activity in the group were significantly increased (*p<0.05). Control There was no obvious calcium nodule formation in the group, but in contrast, CM EV-DOPA The group had significantly more calcium nodules positive staining and higher absorbance values ​​(**p<0.01).

[0111] (2) Osteogenic differentiation indicators

[0112] RT-qPCR test was used to characterize osteogenic differentiation, and the specific implementation was the same as step (3) in Example 4. Figure 29 is a graph of RT-qPCR detection of osteogenic differentiation-related genes under EV-DOPA regulated immune conditions; As can be seen from Figure 29, under the DM microenvironment, the DM microenvironment was significantly different from that of the CM microenvironment. Control Compared with the control group, the gene expression levels of Alp, Runx2, Ocn, and Col1a1 in the EV-DOPA group were significantly increased (*p<0.05, **p<0.01, ***p<0.001). Figure 30 is an image of immunofluorescence staining of Runx2 and OCN, proteins related to osteogenic differentiation; as shown in Figure 30, protein immunofluorescence staining analysis showed that the expression of Alp, Runx2, Ocn, and Col1a1 in the EV-DOPA group was significantly increased compared with the control group (*p<0.05, **p<0.01, ***p<0.001). Control Compared with the CM group, EV-DOPA The expression of Runx2 in the early stage and OCN protein in the middle and late stages increased in the control group (*p<0.05, **p<0.01). The results in this section confirm that EV-DOPA can promote osteogenic differentiation by regulating the immune response mediated by macrophage polarization.

[0113] Example 7: EV-DOPA promotes periprosthetic bone integration in DM rats

[0114] (1) Establishment of animal model

[0115] A streptozotocin (STZ; SigmaAldrich)-induced diabetes model was established. Five-week-old male SD rats weighing 100 g were adaptively fed for 1 week. STZ was added to a citric acid buffer solution with a pH of 4.2-4.5 at a concentration of 1% (w / v) to prepare an STZ solution. After fasting for 12 hours, the rats were injected with STZ at a dose of 35 mg / kg via intraperitoneal injection. Blood from the tail vein of the rats was obtained weekly to analyze the non-fasting blood glucose level (BGL). A BGL > 16.7 mM was confirmed as a successful modeling. A glucose tolerance test (GTT) was further performed to detect pancreatic islet function. The rats were fasted for 12 hours before the test. Blood was collected from the tail vein, and blood glucose was measured as the control value before intraperitoneal injection of glucose solution (1 g / kg). Blood glucose was measured 30, 60, 90, and 120 minutes after injection for comparison to observe the glucose tolerance level. The results showed that the diabetes model had been successfully established.

[0116] Titanium rod implantation was performed 3 weeks after STZ injection modeling. An incision was made in the midline of the rat knee joint to expose the distal femur. A hole was drilled in the center, and titanium rods (diameter: 1.5 mm, length: 10 mm) were implanted into the distal femur parallel to the longitudinal axis of the femur. Animals were divided into four groups based on the different methods of implant material modification: PBS-immersion group (Control), DOPA-modified group (DOPA), EV-simple immersion group (EV), and EV-DOPA-modified group (EV-DOPA).

[0117] During the modeling period, animals in each group were able to move freely within their cages, eat normally, and consume increased water and urine output, with no significant weight gain. There were no significant changes in mental state. Figure 31 shows blood glucose monitoring in DM rats: A. Fasting blood glucose testing at different ages; B. Glucose tolerance testing. As shown in Figure 31, one week after modeling, the rats' blood glucose levels increased significantly. Post-implantation surgery revealed no infection, swelling, exudation, or other inflammatory reactions in the incisions, and all wounds healed in their original form. No animals died during the experiment.

[0118] (2) Early changes in periprosthetic macrophage polarization

[0119] Immunohistofluorescence staining was used to observe the polarization of macrophages around the prosthesis. The specific procedures were as follows:

[0120] 1) Dewaxing and hydration

[0121] Before dewaxing, bake the sections in a 60°C thermostat for 30 minutes. Soak the sections in xylene for 10 minutes, then replace the xylene and soak for another 10 minutes; soak in anhydrous ethanol for 5 minutes; soak in 95% ethanol for 5 minutes; soak in 70% ethanol for 5 minutes;

[0122] 2) Antigen retrieval

[0123] Enzyme digestion method: 0.1% trypsin is commonly used. Trypsin is preheated to 37°C before use, and the slices are also preheated to 37°C. 0.2 ml of digestive solution is added to each slice to completely cover the tissue. Digest in a 37°C incubator for about 5 to 30 minutes, away from light.

[0124] 3) Immunohistofluorescence staining

[0125] Wash 2-3 times with PBS for 5 minutes each; add normal goat serum blocking solution and incubate at room temperature for 20 minutes. Shake off excess liquid; add 100 μl of Antibody I and incubate at 4°C overnight or at 37°C for 1 hour; wash 3 times with PBS for 5 minutes each; add 40-50 μl of Antibody II and incubate at room temperature or at 37°C for 1 hour; wash 3 times with PBS for 5 minutes each; add 20 μl of DAPI-containing anti-fluorescence quenching mounting medium, cover with a coverslip, and observe under a fluorescence microscope.

[0126] Figure 32 is an immunofluorescence co-staining image of CD86 and an analysis of the mean fluorescence intensity; Figure 33 is an immunofluorescence co-staining image of CD206 and an analysis of the mean fluorescence intensity; As can be seen from Figures 32-33, 2 weeks after implantation of the titanium rod prosthesis, the expression level of the M1 macrophage marker protein CD86 in the periprosthetic tissue of the PBS control group was higher, the number of CD68 / CD86 double-positive cells was higher, while the number of cells positive for CD86 / CD206 (M2 macrophages) was lower, and the expression of M2 macrophages was low. In contrast, the expression of CD86 in the EV-DOPA group was significantly reduced, the number of CD68 / CD86-positive M1 macrophages decreased significantly, and the number of CD86 / CD206-positive M2 macrophages increased (*p<0.05, **p<0.01, ***p<0.001). These results confirm that functional modification of the EV-DOPA prosthesis surface can regulate the polarization phenotype of periprosthetic macrophages.

[0127] (3) Micro-CT detection

[0128] The mouse hind limbs were scanned and analyzed using the high-resolution micro-CT SkyScan1076 produced by SkyScan, Belgium. Before scanning, the specimens were removed from the fixative and dried. Each specimen was placed in a Micro-CT test tube cup, 5 at a time, and each specimen was separated by a foam plastic sheet; the specimens should be placed neatly to avoid touching the test tube wall. The scanning parameters were set as follows: voltage 50kV, current 800μA, scanning time 1750ms, spatial resolution 18μm. After the scan was completed, the SkyScan1176 built-in software was used to perform three-dimensional reconstruction of the mouse ankle joint. The following parameters were analyzed using CT Analyzer analysis software: bone density (BMD; mg / cm 3); bone volume fraction (BV / TV; %)); trabecular separation (Tb.Sp; μm) and trabecular number (Tb.N).

[0129] Micro-CT scanning of experimental rat bone tissue, followed by three-dimensional image reconstruction and quantitative analysis, provides a relatively accurate description of bone mass and bone microarchitecture, thereby determining changes in bone parameters and periprosthetic bone integration in rat models. Figure 34 shows three-dimensional reconstruction images of bone mass and bone volume fraction in healthy and DM rats before and after titanium rod implantation. Figure A shows before and after titanium rod implantation. As shown in Figure 34, compared to healthy rats, DM rats exhibit decreased long bone density and impaired periprosthetic bone integration after titanium rod implantation (#p<0.05, ##p<0.01). Bone 3D reconstruction and bone parameter analysis were performed on experimental tissues from different groups. Figure 35 shows a Micro-CT 3D bone reconstruction image demonstrating the effect of EV-DOPA on periprosthetic bone integration in DM rats. Figure 36 shows an analysis of bone volume fraction, bone density, and trabecular thickness, demonstrating the effect of EV-DOPA on periprosthetic bone integration in DM rats. Figures 35 and 36 show that the control group had lower periprosthetic bone mass, including lower bone density (BMD), bone volume fraction (BV / TV), and trabecular number (Tb.N). In the EV-DOPA group, periprosthetic bone density, bone volume fraction, and trabecular number were significantly increased compared to the control group (*p<0.05, **p<0.01, ***p<0.001). These results confirm that EV-DOPA can effectively increase periprosthetic bone mass.

[0130] (4) Histomorphological staining

[0131] H&E hematoxylin-eosin staining and toluidine blue staining were used to observe tissue morphology. The specific steps of H&E hematoxylin-eosin staining are as follows:

[0132] 1) After the femoral tissue specimen is decalcified with EDTA decalcification solution, it is trimmed and embedded in paraffin. The paraffin-embedded specimen is sliced ​​using a tissue slicer to produce paraffin sections with a thickness of 6 μm.

[0133] 2) After paraffin sections were dewaxed with xylene (10 min × 3 times), they were sequentially soaked in 100%, 95%, 90%, and 85% ethanol solutions, followed by water immersion for 5 min each. After rinsing with distilled water for 3 min, the sections were stained with hematoxylin solution for 5 min and rinsed with tap water for 5 min. The sections were then differentiated with 1% hydrochloric acid alcohol solution for 60 s and rinsed with tap water for 1 min. The sections were blued in 10% ammonia solution for 60 s and rinsed with tap water for 1 min. The sections were counterstained with 1% eosin solution for 3 min and rinsed with tap water for 1 min. The sections were then dehydrated with conventional gradient ethanol solutions and xylene, transparentized, and mounted with resin.

[0134] The toluidine blue staining method involves the following steps: After hard tissue sectioning, dilute the toluidine blue stain with water (determine the dilution based on the desired staining intensity based on experimental experience, starting with a 1:100 dilution). Briefly soak the slide in the diluted staining solution, then soak it in water several times, and then press and mount the slide according to the selected method.

[0135] Figure 37 is an H&E staining image of the effect of EV-DOPA on periprosthetic bone integration in DM rats; Figure 38 is a toluidine blue staining image; Figure 39 is an analysis of the annulus fibrosus thickness and bone-prosthesis contact ratio; As shown in Figures 37-39, 8 weeks after prosthesis implantation, tissue H&E staining results showed that both the Control and EV groups had a clear annulus fibrosus; compared with the Control group, the thickness of the periprosthetic annulus fibrosus in the EV-DOPA group was significantly reduced (***p<0.001). Toluidine blue staining results showed that the Control and EV groups had less periprosthetic bone growth. In contrast, the EV-DOPA group had significantly more periprosthetic bone growth, and the ratio of bone tissue to prosthetic surface contact area was significantly increased (***p<0.001). These results confirm that EV-DOPA inhibits the formation of the periprosthetic annulus fibrosus and promotes new bone formation and prosthetic surface bone integration.

[0136] (5) Osteogenesis marker detection

[0137] Calcein fluorescence staining was used to characterize the osteogenic properties of the material. Figure 40 shows calcein fluorescence images demonstrating the effect of EV-DOPA on periprosthetic bone formation in diabetic rats. As shown in Figure 40, the calcein fluorescence results show that in both the control and EV groups, the periprosthetic area of ​​calcein fluorescence is small, with indistinct gaps, indicating poor bone formation. Compared with the control group, the periprosthetic area of ​​the EV-DOPA group exhibited greater calcein fluorescence, with significantly wider gaps. Figure 41 shows immunofluorescence staining images of OCN and COL1A1 proteins. As shown in Figure 41, immunofluorescence staining for the osteogenic differentiation markers OCN and COL1A1 shows that the control group exhibited lower expression of these markers, while the EV-DOPA group showed significantly increased expression of both OCN and COL1A1. These results suggest that EV-DOPA functionalization can effectively promote periprosthetic bone formation in the diabetic microenvironment.

[0138] The data were analyzed using SPSS 11.0 statistical software. Data were expressed as mean ± standard deviation (x ± s). Multiple group comparisons were performed using one-way ANOVA. Pairwise comparisons were performed using LSD and Dunnett's t-tests, provided that the overall variances were equal. A p < 0.05 was considered statistically significant.

[0139] The embodiments described are preferred implementations of the present invention, but the present invention is not limited to the above implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention are within the scope of protection of the present invention.

Claims

1. An exosome-modified medical implant with immune regulation and bone induction functions, characterized in that: The exosome-modified medical implant is modified on the surface of the titanium-based material by mussel-derived adhesion polypeptide and azide exosome surface click chemistry modification to achieve the dual effects of immune regulation and bone induction.

2. The exosome-modified medical implant according to claim 1, characterized in that The exosome-modified medical implant is modified on the titanium-based material surface with a mussel-derived polypeptide (DOPA) that can click-react with the azide groups linked to the exosomes. x ‐PEG5‐DBCO; exosome DSPE-PEG after azidation 2k -Azido and (DOPA) x ‐PEG5‐DBCO achieves stable and lossless modification of exosomes through cycloaddition bioorthogonal click chemistry reaction.

3. A method for preparing an exosome-modified medical implant having dual functions of immune regulation and bone induction, characterized in that: The method comprises the following steps: (1) The solid phase peptide synthesis method was used to synthesize mussel-derived peptides from the copper-free click chemistry reagent dibenzocyclooctyne DBCO, acetone and Fmoc-protected levodopa amino acid Fmoc-DOPA (acetonide)-OH. The obtained mussel-derived peptide was denoted as (DOPA) x ‐PEG5‐DBCO; (2) Immersing the medical implant into the (DOPA) obtained in step (1) by a solution immersion method x -PEG5-DBCO solution, and the DOPA group is fully adhered to the implant surface to obtain a DOPA-implant; (3) MSC-derived exosomes EVs were coupled with the azide coupling reagent 1,2-distearoyl-sn-glycero-3-hydrogenated ethanolamine phosphate DSPE-PEG 2k -Azido mixture, exosome EV combined with DSPE to obtain azidated exosomes, denoted as EV-DSPE-PEG 2k -Azido; (4) Soaking the DOPA-implant obtained in step (2) into the EV-DSPE-PEG obtained in step (3) 2k -Azido solution, the specific loading of exosomes is achieved through cycloaddition bioorthogonal click chemistry reaction between the azide group and the DBCO group, and EV-DOPA-modified exosome-modified medical implants with dual functions of immune regulation and bone induction are obtained.

4. The preparation method according to claim 3, characterized in that: In step (1), the structural formula of the amino acid sequence of the levodopa amino acid is: Ac-[(DOPA)-G] x / 2 -K[(PEG5)‐(Mpa)‐(Mal‐DBCO)]‐[(DOPA)‐G] x / 2 , wherein x is the number of repeating units of DOPA amino acids, and x can be selected from 2, 4, 6 or 8; the overall chain length of the mussel-derived polypeptide is 5, 9, 13 or 17 amino acid groups, and the connection mode is linear.

5. The preparation method according to claim 3, characterized in that: The medical implant described in step (2) can be made of titanium alloy material, preferably a titanium sheet, a titanium rod or a titanium nail.

6. The preparation method according to claim 3, characterized in that: As described in step (2) (DOPA) x The concentration of the PEG5-DBCO solution is 0.005 mg / ml-1.000 mg / ml, and the sufficient soaking time is 12-36 hours.

7. The preparation method according to claim 3, characterized in that: In step (3), EV and DSPE-PEG 2k -The quantity ratio of Azido is 1:100~1:10000.

8. The preparation method according to claim 3, characterized in that: EV-DSPE-PEG described in step (4) 2k The concentration of the Azido solution is 0.01-1.0 mg / mL, and the soaking time is 6-24 hours.

9. Use of the exosome-modified medical implant with dual functions of immune regulation and bone induction according to any one of claims 1-2 in the field of preparing artificial prosthetic implant materials.

10. The use according to claim 9, characterized in that: The exosome-modified medical implant is used in the field of preparing artificial prosthetic implant materials for diabetic patients.

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