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

Abstract

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. Inspired by marine mussels, the present invention combines the metal-phenol coordination chemistry of mussel-like adhesion molecules with the bioorthogonal click chemistry modification strategy to design a mussel-derived adhesion polypeptide with a catechol group. The implant is modified by the catechol / titanium coordination interaction, and it is connected with the azide-modified exosomes, so that the exosomes can be modified on the surface of the medical metal implant material without loss, and the exosome-modified metal bone implant material is obtained, and the bone integration efficiency of the implant material surface is improved by the immunoregulatory function and bone induction activity conferred by it. A new technical method is provided for the efficient modification of functionalized extracellular vesicles on the surface of metal implants, which has a far-reaching impact on improving the survival rate of prosthetic implants in diabetic patients, and has broad prospects in the medical field.

Description

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

[0003] Prosthesis implantation is one of the most successful treatment strategies for bone and joint function reconstruction. However, the immune response after prosthesis implantation will hinder bone integration and affect the effect of bone integration, thus leading to an increase in the failure rate of orthopedic implants. In prosthesis implantation, the biological quality of bones is maintained through the bone remodeling process. Bone metabolism and immune inflammatory response are the main factors affecting osteogenesis during bone remodeling. The characteristics of bone metabolism are mainly concentrated on the weakening of bone formation and the reduction of circulating levels of biochemical markers of bone formation. In addition to bone metabolism, the more important factor affecting osteogenesis is the inflammatory immune response. Studies have shown that inflammatory cytokines released by immune cells can be detected in the body of prosthesis implant recipients, among which macrophages play a key role. When the prosthesis is implanted in the human body, exogenous biomaterials will aggravate the local immune response. Within a few hours after surgery, macrophages will be recruited to the surface of the implant and trigger an early inflammatory cascade. Long-term inflammatory exposure will cause the formation of the surrounding annulus fibrosus, which will develop into a key factor affecting the survival rate of the prosthesis and create a barrier to bone integration. In addition, long-term exposure to this inflammatory environment will also lead to damage to MSC activity and osteogenic differentiation ability. 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 prosthesis 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 prosthesis surface. Summary of the invention

[0005] In order to solve some problems in the prior art, the present invention provides an exosome-modified metal implant with immune regulation and bone induction functions and its preparation method and application. The present invention synthesizes a mussel-derived polypeptide (DOPA) that can adhere to the surface of titanium-based materials and can click with the azide group linked to the exosomes through the bio-clickable group DBCO. x -PEG5-DBCO); the present invention synthesizes a peptide and connects it to a clickable, highly active phospholipid poly (ethylene glycol) derivative 1,2-distearoyl-sn-glycero-3-hydrogenated ethanolamine phosphate azide (DSPE-PEG) pre-differentiated MSC-exosome. 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 the polarization of macrophages in vivo and in vitro, and promote periprosthetic bone integration.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] 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 by mussel-derived adhesion polypeptide and azide exosome surface click chemistry modification to achieve the dual effects of immune regulation and bone induction.

[0008] 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 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.

[0009] In a specific embodiment, the present invention also 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:

[0010] (1) Design and preparation of mussel-derived adhesion peptides; using solid-phase peptide synthesis, copper-free click chemical reagent dibenzocyclooctyne (DBCO), acetone and Fmoc-protected L-DOPA amino acid Fmoc-DOPA (acetonide)-OH were used to synthesize mussel-derived peptides. The obtained mussel-derived peptide was denoted as (DOPA) x -PEG5-DBCO;

[0011] (2) Derivatization of the surface of medical implants with peptides: The medical implants are immersed in 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;

[0012] (3) Azide modification of exosome surface:

[0013] MSC-derived exosomes (EVs) were conjugated with the azide-coupled reagent 1,2-distearoyl-sn-glycero-3-hydroethanolamine phosphate (DSPE-PEG 2k -Azido) was mixed, and the exosomes EV were combined with DSPE to obtain azidated exosomes, which were recorded as EV-DSPE-PEG 2k -Azido;

[0014] (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, 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.

[0015] 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.

[0016] 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).

[0017] During the synthesis of the mussel-derived adhesion polypeptide in step (1), the dopa groups inside a single polypeptide molecule are spaced apart 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.

[0018] The invention uses acetone as a protecting agent to protect the catechol group in levodopa amino acid (DOPA).

[0019] The medical implant described in step (2) can be selected from titanium alloy materials, preferably titanium sheets, titanium rods or titanium nails. 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.

[0020] In step (3), EV and DSPE-PEG 2k -The quantity ratio of Azido is 1:100~1:10000.

[0021] 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.

[0022] 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.

[0023] Preferably, the exosome-modified medical implant is used in the field of preparing artificial prosthetic implant materials for diabetic patients.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] Inspired by marine mussels, the present invention combines the metal-phenol coordination chemistry of mussel-like adhesion molecules with the bioorthogonal click chemistry modification strategy to design a mussel-derived adhesion polypeptide with a catechol group. The metal Ti implant is modified through a strong catechol / titanium (Ti) coordination interaction, and it is connected with the azide-modified exosomes to obtain an exosome-modified medical implant with dual functions of immune regulation and bone induction. The exosomes are non-destructively modified on the surface of the medical metal implant material, giving the implant material immune regulation function and bone induction activity.

[0026] The present invention modifies the surface of bone implants with exosomes from mesenchymal stem cells that have multiple biological activities and early osteogenic differentiation by imitating the mussel adhesion mechanism and bio-orthogonal click reaction. It can regulate the polarization phenotype of macrophages, exert the regulatory effect of immune-osteogenesis cascade, and thus improve the performance of implants. It can reduce the inflammatory response under the hyperglycemic microenvironment, reduce the expression of IL-1β, IL-6 and TNF-α related inflammatory factors; promote the osteogenic differentiation ability of BMSC under the hyperglycemic microenvironment; promote periprosthetic bone integration under DM environment, promote bone formation, and increase bone density; the obtained exosome-modified medical implant has good biocompatibility, promotes the polarization of macrophages to the anti-inflammatory M2 phenotype, and synergistically improves the immune microenvironment induction of the bone-implant interface and the optimal osteogenic performance and bone integration effect of the bone-implant interface. Compared with the complexity, potential toxicity and destructiveness of traditional chemical modification, the mussel-like adhesion and click modification strategy involved in the present invention has a fast and mild preparation method that does not destroy the structural stability of exosomes, providing a new method for efficient modification of functionalized exosomes on the surface of medical metal implants. It has a profound impact on improving the survival rate of diabetic patients with implanted prostheses and has broad prospects in the medical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the flow cytometry identification diagram of primary MSC markers;

[0028] 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;

[0029] Figure 3 This is a schematic diagram of the molecular structure of the components of mussel-derived peptides; in the figure, a is the representative molecular formula of mussel-derived peptide (DOPA) 6-PEG5-DBCO, and b is DSPE-PEG 2k -Azido molecular formula, c is the cycloaddition reaction formula;

[0030] Figure 4 It is a representative HPLC analysis and MS analysis chart of (DOPA)6-PEG5-DBCO;

[0031] Figure 5 DSPE-PEG 2k -NMR analysis of Azido;

[0032] Figure 6 It is the image directly observed by FITC fluorescence microscope;

[0033] Figure 7 It is the surface morphology detection image of atomic force microscope (AFM);

[0034] Figure 8 It is the X-ray photoelectron spectroscopy (XPS) surface element composition detection map;

[0035] Fig. 9 It is the water contact angle test (WCA) graph;

[0036] Fig.10 is the SEM image of MSC and RAW 264.7 cells;

[0037] Fig.11 It is the average cell spreading area analysis and cell proliferation activity detection graph;

[0038] Fig.12 are cell live-dead staining images (**p<0.01);

[0039] Fig.13 It is the fluorescence image of reactive oxygen species detection by EV-DOPA’s direct osteogenic effect;

[0040] Fig.14 This is the flow cytometry quantitative diagram of the reactive oxygen species detection of EV-DOPA's direct osteogenic effect;

[0041] Fig.15 This is the staining diagram of the direct osteogenic effect of EV-DOPA: In the figure, A is ALP staining; B is ARS staining;

[0042] Fig.16 This is the ALP activity detection chart and ARS absorbance detection chart of EV-DOPA's direct osteogenic effect;

[0043] Fig.17 This is the RT-qPCR detection chart of genes related to osteogenic differentiation in which EV-DOPA directly promotes osteogenesis;

[0044] Fig.18 This is the result of immunofluorescence staining of related proteins Runx2 and OCN;

[0045] Fig.19 This is a schematic diagram of the experiment of EV-DOPA regulating the polarization phenotype change of macrophages;

[0046] Fig. 20 This is the co-staining image of iNOS and F4 / 80 immunofluorescence in macrophages;

[0047] Fig.21 is the immunofluorescence co-staining image of macrophage Arg-1 and F4 / 80;

[0048] Fig. 22 is the relative mean fluorescence intensity analysis graph of immunofluorescence staining;

[0049] Fig.23 This is a quantitative flow cytometry analysis of CD86, a macrophage M1 polarization phenotype;

[0050] Fig.24 This is a quantitative flow cytometry analysis of CD206, a macrophage M2 polarization phenotype;

[0051] Fig.25 This is the RT-qPCR detection graph of inflammatory cytokines;

[0052] Fig.26 This is a high-throughput sequencing analysis of inflammatory factor expression;

[0053] Fig. 27 These are the staining images under EV-DOPA regulated immune conditions: In the figure, A is ALP staining; B is ARS staining;

[0054] Fig.28 This is the ALP positive staining area and ARS staining absorbance detection diagram under EV-DOPA regulated immune conditions;

[0055] Fig.29 This is the RT-qPCR detection graph of osteogenic differentiation-related genes under EV-DOPA-regulated immune conditions;

[0056] Fig.30 This is an immunofluorescence staining image of osteogenic differentiation-related proteins Runx2 and OCN;

[0057] Fig.31 This is the blood glucose monitoring chart of DM rat modeling: A fasting blood glucose test at each age stage; B glucose tolerance test;

[0058] Fig.32 is the CD86 immunofluorescence co-staining image and mean fluorescence intensity analysis graph;

[0059] Fig.33 is the CD206 immunofluorescence co-staining image and mean fluorescence intensity analysis graph;

[0060] Fig.34 The three-dimensional reconstruction images and bone volume fraction analysis diagrams of healthy rats and DM rats before and after titanium rod implantation in the femur; in the figure, A is before titanium rod implantation, and B is after titanium rod implantation;

[0061] Fig.35 This is a Micro-CT bone three-dimensional reconstruction image of the effect of EV-DOPA on periprosthetic bone integration in DM rats;

[0062] Fig.36 The bone volume fraction analysis, bone density analysis and trabecular thickness analysis of the effect of EV-DOPA on periprosthetic bone integration in DM rats;

[0063] Fig.37 is the H&E staining image of the effect of EV-DOPA on periprosthetic bone integration in DM rats;

[0064] Fig.38 is a toluidine blue staining image;

[0065] Fig.39 It is the analysis diagram of the annulus fibrosus thickness and the bone-prosthesis contact ratio;

[0066] Fig.40 It is the calcein fluorescence image of the effect of EV-DOPA on periprosthetic bone formation indices in DM rats;

[0067] Fig.41 These are images of immunofluorescence staining of OCN and COL1A1 proteins. DETAILED DESCRIPTION

[0068] The present invention can be further described by the following examples, however, the scope of the present invention is not limited to the following examples. The present invention generally and / or specifically describes the materials and test methods used in the experiments. The experimental methods in the following examples without specifying specific conditions are usually tested under conventional conditions or under conditions recommended by the manufacturer. The reagents, biological materials, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[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, PrimeScriptRTMaster Mix, Ex Taq TM The primary antibody working solution, secondary antibody working solution, goat serum blocking solution, I antibody and II antibody were purchased from Abcam. SD rats were 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; using solid-phase peptide synthesis, copper-free click chemical reagent dibenzocyclooctyne (DBCO), acetone and Fmoc-protected L-DOPA amino acid Fmoc-DOPA (acetonide)-OH were used to synthesize mussel-derived peptides. The obtained mussel-derived peptide was denoted 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 are immersed in the (DOPA) obtained in step (1) by a solution immersion method. x -PEG5-DBCO solution (concentration of 0.005mg / ml-1.000mg / 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 mixed with 100- to 10,000-fold amounts of the azide coupling reagent 1,2-distearoyl-sn-glycero-3-hydrogenated ethanolamine phosphate (DSPE-PEG 2k -Azido) was mixed, and the exosomes EV were combined with DSPE to obtain azidated exosomes, which were 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 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.

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

[0077] 6-8 week old SD rats (weight 200g, SPF grade, Soochow University Animal Experiment Center) were purchased and anesthetized to extract primary bone marrow mesenchymal stem cells (BMSC) from long bones. After culturing for 3 days in osteogenic induction medium containing exosome-free serum, the supernatant was extracted and centrifuged to remove cell debris, etc., and then exosomes were extracted by ultracentrifugation. NTA particle size analysis and protein marker detection were used to detect exosomes.

[0078] Figure 1 This is the 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; Figure 1-Figure 2 It can be seen that the results of flow cytometry identification showed that MSCs cells were CD34, CD45 negative and CD29, CD90 positive; the results of NTA particle size analysis showed that the diameter distribution of the extracted microvesicles was concentrated in 100-200nm; the exosome markers CD9, CD63, and TSG101 were tested positive, indicating that the exosomes of primary bone marrow mesenchymal stem cells had been successfully obtained.

[0079] Example 2: Preparation of EV-DOPA coated implants

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

[0081] Acetone-protected Fmoc-DOPA (acetonide)-OH was used to synthesize mussel-derived peptides by solid-phase peptide synthesis. The 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 L-DOPA 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 This is a schematic diagram of the molecular structure of the components of mussel-derived peptides; in the figure, a is the representative molecular formula of mussel-derived peptide (DOPA) 6-PEG5-DBCO, 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; Figure 4It can be seen that 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] The medical titanium sheet was immersed in a (DOPA)6-PEG5-DBCO solution (0.005-1.000 mg / ml) for 24 hours to allow the DOPA group to fully adhere to the titanium surface. The free peptide that could not be grafted on the surface was washed away with ultrapure water to obtain a polypeptide-treated implant, which was recorded as DOPA-DBCO-Ti.

[0084] (3) Azidation 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 -Azido. Figure 5 is the NMR analysis chart of DSPE-PEG2k-Azido; Figure 5 Nuclear magnetic resonance spectroscopy (NMR) analysis showed that DSPE-PEG 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 the titanium-based material after DOPA adhesion was fully click-connected for 12 hours. The specific loading of exosomes was achieved through the cycloaddition bioorthogonal click chemistry reaction between azide and diphenylcyclooctyne (DBCO) groups, and an exosome-modified medical implant material with dual functions of immune regulation and bone induction (EV-DOPA) was obtained, which was recorded as 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 embodiment 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 visible fluorescent groups 2k-FITC was dissolved in sterile PBS at a ratio of 3:1 (w:w) to form a mixed solution, and then the exosomes derived from MSCs during osteogenic differentiation were mixed with the mixed solution at a molar concentration of 1:1000. After full fusion, azide-modified EVs were obtained, which were recorded as FITC / (2-Azido)-PEG. 2k -DSPE-EV; FITC / (2-Azido)-PEG 2k -DSPE-EV reacts with DOPA-DBCO-Ti to obtain a labelable exosome-modified medical implant material with dual functions of immune regulation and bone induction, which is recorded 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 observed under a fluorescence microscope. Among them, the Control group was a titanium sheet soaked in ordinary PBS, the EV group was a titanium sheet modified only with exosomes, and the DOPA group was a titanium sheet modified only with DOPA groups obtained in step (2) of Example 1. Figure 6 It is the image directly observed by FITC fluorescence microscope; Figure 6 It can be seen that green dot-like fluorescence was observed on the titanium surface of FITC-EV-DOPA carrying FITC groups, while no fluorescence was observed in other groups. Figure 7 It is the surface morphology detection image of atomic force microscope (AFM); Figure 7 It can be seen that compared with the titanium sheet soaked in ordinary PBS, the surface roughness of the DOPA group increased, there was no obvious change in the EV group, and a large number of exosome spherical protrusions were observed in the EV-DOPA group. Figure 8 It is the X-ray photoelectron spectroscopy (XPS) surface element composition detection map; Figure 8 It can be seen that compared with the titanium sheet soaked in ordinary PBS, the content of "N" element on the surface of the modified titanium-based material is significantly increased; Fig. 9 is the water contact angle test (WCA) diagram; Fig. 9 It can be seen that compared with the titanium sheet soaked in ordinary PBS, the water contact angle of the EV-DOPA group was significantly reduced, the hydrophilicity was better, and the EV was effectively modified on the surface of the titanium alloy without destroying the overall structure of the EV.

[0092] MSCs and RAW264.7 cells (homemade) were seeded on the titanium surface of each group. Fig.10 is the SEM image of MSC and RAW 264.7 cells; Fig.11It is the average cell spreading area analysis and cell proliferation activity detection graph; Fig.12 is the live-death staining image of cells (**p<0.01); Figure 10-12 It can be seen that the SEM images showed 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 cell proliferation detection showed that compared with the Control group, the cell proliferation activity in the EV-DOPA group was higher. 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 performance

[0094] (1) Antioxidant effect of EV-DOPA

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

[0096] Fig.13 It is the fluorescence image of reactive oxygen species detection by the direct osteogenic effect of EV-DOPA; Fig.14 It is the quantitative flow chart of reactive oxygen species detection by EV-DOPA directly promoting osteogenic effect; Fig.13 , Fig.14 It can be seen that the results of reactive oxygen fluorescence and flow cytometry detection showed that under the DM microenvironment, more reactive oxygen species were produced in the Control group and the EV group. Compared with the Control group, the reactive oxygen species produced in the BMSCs of the EV-DOPA group were significantly reduced.

[0097] (2) ALP and ARS staining

[0098] Primary MSC cells were inoculated in the Control group, EV group, DOPA group and EV-DOPA group, 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 stained with ALP alkaline phosphatase staining kit, and images were collected under an optical microscope. Positively stained cells were 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. Fig.15This is the staining diagram of the direct osteogenic effect of EV-DOPA: In the figure, A is ALP staining; B is ARS staining; Fig.16 It is the ALP activity detection chart and ARS absorbance detection chart of EV-DOPA's direct osteogenic effect; Figure 15-16 It can be seen that the positively stained calcium nodules are red, the ALP level in the Control group is low, and compared with the Control group, the oxidative stress level of MSC cells in the EV-DOPA group is significantly reduced, and the ALP positive staining area and ALP activity are significantly increased (***p<0.001). There is no obvious calcium nodule formation in the Control group, while in comparison, the EV-DOPA group has 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 by NanoDrop-2000. PrimeScriptRT Master Mix was used for reverse transcription. SYBR1 Premix Ex Taq 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] The results of gene and protein detection of osteogenic differentiation-related indicators showed that EV-DOPA modification promoted the expression of osteogenic-related genes (Alp, Runx2, Ocn, Col1a1, Sp7) and the protein expression of Runx2, an early osteogenic differentiation indicator, and OCN, a mid- to late-stage indicator. RAW264.7 cells were then inoculated on the surface of titanium sheets in different groups and cultured in a DM simulated microenvironment. Fig.17 This is the RT-qPCR detection chart of genes related to osteogenic differentiation in which EV-DOPA directly promotes osteogenesis; Fig.18 This is the result of immunofluorescence staining of related proteins Runx2 and OCN; Figure 17-Figure 18It can be seen that the RT-qPCR results showed that the expression of proinflammatory cytokines in the EV-DOPA modified group was lower than that in the control group. Under the DM microenvironment, compared with the Control group, the gene expression levels of Alp, Runx2, Ocn, Col1a1 and Sp7 in the EV-DOPA group were significantly increased (*p<0.05, **p<0.01, ***p<0.001). By protein immunofluorescence staining analysis, compared with the Control group, the expression of early Runx2 and mid-late OCN proteins in the EV-DOPA group increased (*p<0.05, **p<0.01, ***p<0.001). The results of this part confirmed that EV-DOPA modification inhibited the differentiation of macrophages into proinflammatory M1 type and increased the number of anti-inflammatory M2 type macrophages. EV-DOPA has direct antioxidant and osteogenic differentiation functions.

[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. Fig.19 This is a schematic diagram of the experiment on EV-DOPA regulating the polarization phenotype transformation of macrophages. 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] Fig. 20 This is the co-staining image of iNOS and F4 / 80 immunofluorescence in macrophages; Fig.21 is the immunofluorescence co-staining image of macrophage Arg-1 and F4 / 80; Fig. 22 is the relative mean fluorescence intensity analysis diagram of immunofluorescence staining; Figure 20-22 It can be seen that the results of cell immunofluorescence staining showed that in the DM microenvironment, the expression of iNOS in macrophages was higher, while the expression of Arg-1 was lower; compared with the Control group, the expression of iNOS in the EV-DOPA group cells was reduced, while the expression of Arg-1 was increased. Fig.23 This is a quantitative flow cytometry analysis of CD86, a macrophage M1 polarization phenotype; Fig.24 is a quantitative flow cytometry analysis of CD206, a macrophage M2 polarization phenotype; Figure 23-Figure 24It can be seen that 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. Fig.25 is the RT-qPCR detection chart of inflammatory cytokines; Fig.25 It can be seen that the RT-qPCR results showed that in 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 decreased, while the anti-inflammatory IL-10 gene expression was increased (*p<0.05, **p<0.01, ***p<0.001). Fig.26 is the analysis chart of inflammatory factor expression by high-throughput sequencing; Fig.26 It can be seen that through the analysis of high-throughput sequencing results, EV-DOPA can significantly reduce the expression of most inflammatory-related factors in 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 procedures were the same as step (2) in Example 4. After the EV-DOPA-modified prosthesis was co-cultured with the conditioned medium prepared by macrophage culture, Fig. 27 These are the staining images under EV-DOPA regulated immune conditions: In the figure, A is ALP staining; B is ARS staining; Fig.28 It is the ALP positive staining area and ARS staining absorbance detection diagram under EV-DOPA regulated immune conditions; Fig. 27 , Fig.28 It can be seen that 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 CM 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 nodule 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. Fig.29 This is the RT-qPCR detection diagram of osteogenic differentiation-related genes under EV-DOPA regulated immune conditions; Fig.29 It can be seen that in the DM 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). Fig.30 This is an immunofluorescence staining image of osteogenic differentiation-related proteins Runx2 and OCN; Fig.30 It can be seen that protein immunofluorescence staining analysis and CM Control Compared with the CM group EV-DOPA The expression of Runx2 in the early stage and OCN protein in the middle and late stage increased in the two groups (*p<0.05, **p<0.01). The results in this part all confirmed 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; Sigma Aldrich)-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 with a pH of 4.2-4.5 at a concentration of 1% (w / v) to prepare an STZ solution. After the rats were fasted for 12 hours, STZ was injected intraperitoneally at a dose of 35 mg / kg. Blood from the tail vein of the rats was obtained weekly to analyze the non-fasting blood glucose level (BGL), and 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 at 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 opening 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) of each group were implanted into the distal femur parallel to the longitudinal axis of the femur. According to the different ways of implant material modification, the animals were divided into 4 groups: PBS soaked group (Control), DOPA modified group (DOPA), EV simple soaked group (EV) and EV-DOPA modified group (EV-DOPA).

[0117] During the modeling period, the animals in each group were free to move in the cage, eat normally, have increased water intake and urine output, but no significant increase in body weight or mental state. Fig.31 This is the blood glucose monitoring chart of DM rat modeling: A fasting blood glucose test at each age; B glucose tolerance test; Fig.31 It can be seen that one week after modeling, the blood sugar of rats increased significantly. There was no infection or inflammatory reaction such as swelling, exudation, etc. after the implantation, and all healed in the first stage. No animals died during the experiment.

[0118] (2) Early changes in polarization of periprosthetic macrophages

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

[0120] 1) Dewaxing and hydration

[0121] Before dewaxing, bake the slices in a 60℃ thermostat for 30 minutes. Soak the slices in xylene for 10 minutes, then replace 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. Add 0.2 ml of digestion solution 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 with PBS 2-3 times for 5 minutes each; add normal goat serum blocking solution and incubate at room temperature for 20 minutes. Shake off excess liquid; add 100μl of I antibody and incubate at 4℃ overnight or at 37℃ for 1 hour; wash with PBS 3 times for 5 minutes each; add 40-50μl of II antibody and incubate at room temperature or at 37℃ for 1 hour; wash with PBS 3 times for 5 minutes each; add 20μl of DAPI anti-fluorescence quenching sealing agent, cover the sealing slide with a coverslip, and observe under a fluorescence microscope.

[0126] Fig.32 is the CD86 immunofluorescence co-staining image and mean fluorescence intensity analysis graph; Fig.33 is the CD206 immunofluorescence co-staining image and average fluorescence intensity analysis diagram; Figure 32-Figure 33 It can be seen that 2 weeks after titanium rod prosthesis implantation, the expression level of 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 more, and the number of CD86 / CD206 positive cells (M2 macrophages) was less, 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 EV-DOPA prosthesis surface functional modification can regulate the polarized phenotype of periprosthetic macrophages.

[0127] (3) Micro-CT testing

[0128] The mouse hind limbs were scanned and analyzed using the high-resolution micro-CT SkyScan 1076 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] Using Micro-CT to scan the bone tissue of experimental rats and perform three-dimensional image reconstruction and quantitative analysis can more accurately describe the bone mass and bone microstructure, thereby determining the changes in bone parameters of the rat model and the bone integration around the prosthesis. Fig.34 The three-dimensional reconstruction images and bone volume fraction analysis diagrams of healthy rats and DM rats before and after titanium rod implantation in the femur; in the figure, A is before titanium rod implantation, and B is after titanium rod implantation; Fig.34 It can be seen that compared with healthy rats, the bone density of long bones in DM model rats decreased, and the bone integration ability around the prosthesis was insufficient after titanium rod prosthesis implantation (#p<0.05, ##p<0.01). Bone three-dimensional reconstruction and bone parameter analysis were performed on experimental tissues in different groups. Fig.35This is a Micro-CT bone three-dimensional reconstruction image of the effect of EV-DOPA on periprosthetic bone integration in DM rats; Fig.36 The figure is the bone volume fraction analysis, bone density analysis and trabecular thickness analysis of the effect of EV-DOPA on periprosthetic bone integration in DM rats; Figure 35-Figure 36 It can be seen that the bone mass around the prosthesis was low in the control group, and the bone density (BMD), bone volume fraction (BV / TV) and trabecular number (Tb.N) were all at low levels; in the EV-DOPA group, the bone density, bone volume fraction and trabecular number around the prosthesis were significantly increased compared with the Control group (*p<0.05, **p<0.01, ***p<0.001). These results confirm that EV-DOPA can effectively increase the bone mass around the prosthesis.

[0130] (4) Histomorphological staining

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

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

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

[0134] The steps of the toluidine blue staining method are as follows: After the tissue specimen is sliced, dilute the toluidine blue staining solution with water (the dilution is determined based on the desired staining degree based on experimental experience, starting from a 1:100 dilution). Soak the slide in the diluted staining solution for a short time, then soak it in water several times, and press and fix it according to the selected method.

[0135] Fig.37 is the H&E staining image of the effect of EV-DOPA on periprosthetic bone integration in DM rats; Fig.38 is a toluidine blue staining image; Fig.39 It is the analysis diagram of the annulus fibrosus thickness and the bone-prosthesis contact ratio; Figure 37-Figure 39It can be seen that 8 weeks after prosthesis implantation, the results of tissue H&E staining showed that both the Control group and the EV group had obvious annulus fibrosus; compared with the Control group, the thickness of the annulus fibrosus around the prosthesis in the EV-DOPA group was significantly reduced (***p<0.001). The results of toluidine blue staining showed that the amount of new bone around the prosthesis in the Control group and the EV group was less. In comparison, the new bone around the prosthesis in the EV-DOPA group increased significantly, and at the same time, the contact area ratio between bone tissue and the prosthesis surface increased significantly (***p<0.001). These results confirm that EV-DOPA inhibits the formation of annulus fibrosus around the prosthesis and promotes new bone formation and bone integration of the prosthesis surface.

[0136] (5) Osteogenesis marker detection

[0137] Calcein fluorescence staining was used to characterize the osteogenic properties of the material. Fig.40 is the calcein fluorescence image of the effect of EV-DOPA on periprosthetic bone formation indices in DM rats; Fig.40 It can be seen that the calcein fluorescence results showed that in the Control group and the EV group, the calcein fluorescence area around the prosthesis was small and the gap was not obvious, indicating low bone formation; compared with the Control group, more calcein fluorescence appeared around the prosthesis in the EV-DOPA group, and the fluorescence gap was significantly widened. Fig.41 This is an immunofluorescence staining image of OCN and COL1A1 proteins. Fig.41 It can be seen that according to the results of immunohistofluorescence staining of osteogenic differentiation indicators OCN and COL1A1, the expression of osteogenic indicator proteins in the Control group was low. In contrast, the expression of OCN and COL1A1 in the EV-DOPA group was significantly increased. These results suggest that EV-DOPA functional modification can effectively promote osteogenic differentiation around the prosthesis in the DM microenvironment.

[0138] The results of the present invention were analyzed using SPSS11.0 statistical software, and the data were expressed as mean ± standard deviation (x ± s). One-way ANOVA test was used for multiple group comparisons, and LSD and Dunnett-t methods were used for pairwise comparisons under the condition of homogeneity of overall variance. p < 0.05 was considered statistically significant.

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

Claims

1. 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 chemical 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; 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 polypeptide is 5, 9, 13 or 17 amino acid groups, and the connection mode is linear; K is lysine, and G is glycine; (2) soaking the medical implant in the (DOPA)x-PEG5-DBCO solution obtained in step (1) by a solution immersion method, so that the DOPA group is fully adhered to the surface of the implant to obtain a DOPA-implant; the (DOPA) x - The concentration of the PEG5-DBCO solution is 0.005 mg / ml-1.000 mg / ml, and the sufficient immersion time is 12-36 hours; (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; the EV and DSPE-PEG 2k -The quantity ratio of Azido is 1:100~1:10000; (4) The DOPA-implant obtained in step (2) is immersed in the EV-DSPE-PEG2k-Azido solution obtained in step (3), and the specific loading of exosomes is achieved through a cycloaddition bioorthogonal click chemistry reaction between the azide group and the DBCO group, thereby obtaining an EV-DOPA-modified exosome-modified medical implant with dual functions of immune regulation and bone induction.

2. The preparation method according to claim 1, characterized in that: The medical implant described in step (2) is made of titanium alloy material.

3. The preparation method according to claim 2, characterized in that: The medical implant is a titanium sheet, a titanium rod or a titanium nail.

4. The preparation method according to claim 1, 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.

5. The exosome-modified medical implant with immune regulation and bone induction function prepared by the method according to any one of claims 1 to 4, 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.

6. The exosome-modified medical implant according to claim 5, 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.

7. Use of the exosome-modified medical implant with dual functions of immune regulation and bone induction according to any one of claims 5 to 6 in the field of preparing artificial prosthetic implant materials.

8. The use according to claim 7, 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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