CN114767342A

CN114767342A - Preparation method of bone defect repair stent

Info

Publication number: CN114767342A
Application number: CN202210264291.XA
Authority: CN
Inventors: 姜文彬; 孙家明; 陈雳风; 汪振星; 刘剑; 张郭; 陈宇轩; 詹怡辰; 张一帆
Original assignee: Tongji Medical College of Huazhong University of Science and Technology
Current assignee: Tongji Medical College of Huazhong University of Science and Technology
Priority date: 2022-03-17
Filing date: 2022-03-17
Publication date: 2022-07-22

Abstract

The invention provides a preparation method of a bone defect repair bracket, which comprises the steps of obtaining imaging data of a bone defect part through modes of clinical imaging CT, MRI scanning and the like, then utilizing CAD software to carry out modeling according to the specific shape of the bone defect of a patient, outputting an STL format file of the bone tissue repair bracket, using biodegradable material photocuring polycaprolactone as a printing raw material, utilizing a photoetching 3D printer to stack and mold the material, and printing a porous bone repair bracket suitable for the shape of the defect part of the patient. And then, grafting the biotinylated vesicle on the surface of the scaffold by using a biotin-avidin system to construct the bioactive scaffold with the stem cell vesicle loaded on the surface. According to the invention, the stem cell vesicles are loaded on the porous bone scaffold printed by photocuring by using a biotin-avidin system, so that the scaffold has the effect of accelerating bone defect repair by using active substances in the stem cell vesicles while realizing personalized customization of the bone scaffold.

Description

Preparation method of bone defect repair stent

Technical Field

The invention relates to the field of tissue engineering bone defect repair, in particular to a preparation method of a bone defect repair bracket.

Background

Bone deficiency caused by trauma or surgery is called bone defect, such as comminuted fracture, open fracture large bone tissue defect caused by trauma, inflammation, bone disease and other factors in pathological process, osteonecrosis and abscission separation caused by inflammation, and defect caused by osteonecrosis caused by bone infarction or osteoischemic necrosis. Due to the existence of bone defects, the nonunion of the bone, delayed healing or nonhealing and local dysfunction are often caused; therefore, the repair and functional reconstruction of segmental bone defects caused by trauma and diseases are problems to be solved urgently in clinical orthopedics, and the repair of segmental bone defects comprises autologous bones, allogeneic bones, tissue engineering bone transplantation, artificial bone replacement and the like.

The autologous bone has the defects of limited sources and additional surgical wounds, the allogeneic bone needs to be subjected to freeze-drying and deep freezing treatment, the osteogenesis performance is reduced, and the clinical application of tissue engineering bone is not mature, so that the current artificial bone is widely applied clinically.

However, when the artificial bone repairs segmental bone defects, cells and tissue fluid are difficult to permeate into the artificial bone, the speed of blood vessel growth is quite slow, the bracket material with poor osteogenic activity is easy to be occupied by fibrous tissues in pores, migration and proliferation of osteoblasts are hindered, bone nonunion is easy to occur, and the treatment effect is further influenced;

segmental bone defects are usually caused by serious trauma or tumor resection, so that a greater risk of infection exists, and the existing artificial bone products cannot play the treatment and antibacterial functions, so that the infection cannot be avoided;

the performance of the prosthesis directly affects the personal safety of a patient, and the existing evaluation method cannot accurately and comprehensively evaluate the performance of the prosthesis and has potential risks.

How to improve the vascularization promotion and osteogenesis capacity of the bone repair material is a difficult problem of bone tissue repair. Research finds that the structural design of the bone repair material is the key to influence the vascularization of bone regeneration. Most of the currently adopted bone repair materials have the following two defects: 1. the mechanical property is poor; 2. lack of biological activity.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for manufacturing a bone defect repairing scaffold, which adopts photoetching printing materials with good biocompatibility, the materials can achieve higher elastic modulus through the structural design, and then the surfaces of the materials are grafted with adipose-derived stem cell exosomes of patients by utilizing a biotin-avidin system, so that the biological activity of the bone repairing scaffold can be enhanced, and the function of promoting vascularization and the capability of promoting bone can be improved.

The technical scheme provided by the invention is as follows: a preparation method of a bone defect repair bracket comprises the following steps:

(1) obtaining the imaging data of the bone defect part of the patient in clinical imaging CT and MRI scanning modes, and then performing three-dimensional modeling by using CAD software to obtain a sheet STL format file;

(2) based on the data analysis and the model making, designing an individualized bone repair bracket with a specific shape suitable for the bone defect part of the patient;

(3) dissolving a photoinitiator TPO-L in a concentration of 1.0-2.0% w/v and a light absorbent beta-carotene in a concentration of 0.05-0.1% w/v in photocuring Polycaprolactone (PCLMA) to obtain liquid ink for printing a bone scaffold;

(4) performing sterile treatment (filtering by using a 0.22-micron sterile filter) on the bone scaffold printing liquid ink obtained in the step (3), adding a centrifuge tube and a liquid transfer gun into the bone scaffold printing liquid ink, repeatedly blowing and uniformly mixing the bone scaffold printing liquid ink and the liquid transfer gun, placing the bone scaffold printing liquid ink into the centrifuge tube and the liquid transfer gun for 3000-4000r/min, centrifuging the bone scaffold printing liquid ink for 3-5 minutes to remove bubbles, and heating the bone scaffold printing liquid ink to 37-37.5 ℃ in a water bath kettle for later use;

(5) and (3) carrying out layered slicing processing on the model obtained in the step (2) (because the photoetching printer prints layer by layer, the model is processed into an STL format suitable for printing, namely, the model is cut into a plurality of layers, and the layers are printed and overlapped to form), putting the printing raw materials obtained in the step (4) into a photocuring 3D printer together for aseptic printing, and printing the layers into a solid bone defect repairing support with a porous structure.

(6) And (3) carrying out surface modification treatment on the porous bone defect repair scaffold obtained in the step (5), firstly placing the porous bone defect repair scaffold in a mixed solution of 0.20-0.25mol/L potassium permanganate and 0.4-0.5mol/L hydrochloric acid, carrying out sterile treatment (filtering by a 0.22um sterile filtration membrane) on the mixed solution in advance, oscillating for 8-9 hours at constant temperature of 45-48 ℃ and 150r/min with 120-48 ℃ in a shaking table, exposing partial hydroxyl on the surface of the scaffold by the treatment, and then grafting biotin on the surface of the scaffold by using esterification reaction of the biotin carboxyl so as to uniformly biotinylate the surface of the scaffold.

(7) Biotinylation treatment is carried out on the porous bone repair scaffold with the surface exposed with hydroxyl group obtained in the step (6), 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) is dissolved in anhydrous dichloromethane at the concentration of 0.3% -0.4% w/v, 4-Dimethylaminopyridine (DMAP) is dissolved in the concentration of 0.005% -0.007% w/v, and Biotin (Biotin) is dissolved in the concentration of 0.4% -0.5% w/v to prepare the biotinylation working solution of the bone repair scaffold.

(8) And (4) performing sterile treatment on the working solution obtained in the step (7), (filtering by using a sterile filter membrane with the diameter of 0.22 um), placing the bone repair scaffold obtained in the step (6) in the working solution, and shaking by using a constant temperature shaking table at 47-48 ℃ for 12-14h with the speed of 120-micron and 150 r/mm to obtain the bone repair scaffold with the biotinylation surface.

(9) And (3) placing the biotinylated stent obtained in the step (8) in a culture medium containing biotinylated exosomes, adding streptavidin according to the ratio of 20-25ug/ml of the culture medium, and placing for 30-45min at 37-37.5 ℃, so that the biotinylated exosomes can be tightly combined with the biotinylated stent by utilizing a biotin-avidin system.

Further, the parameters of the 3D printing in the step (5) are: the layer height is 20-25 μm, and the light intensity is 15-20mW/cm²10-12 layers of base layer, base layerThe exposure time is 10-15s, the lamella exposure time is 8-13s, the peeling distance is 6-8mm, the peeling speed is 18-20mm/min, the peeling recovery speed is 100-120mm/min, and the light source wavelength is 405 (specific) nm.

Further, the exosome in the step (9) is autologous adipose-derived stem cell exosome of the patient, adipose tissues of the patient are obtained by a suction method (after the patient is clinically general anesthetized, the patient is firstly subjected to liposuction by using a sterile liposuction needle and a 20ml syringe on an operating table, the abdomen is selected for more parts), the adipose-derived stem cells are digested and extracted by lipase, supernatant is collected during each liquid change, the supernatant is centrifuged for 2 hours at 100000G in an ultracentrifuge, bottom vesicles are collected, and the cells are stored at-80 ℃ for later use. The related Micro RNA and protein in the exosome can promote angiogenesis and osteogenesis, are favorable for the growth of blood vessels in the bone defect support, and further accelerate the regeneration of bone tissues.

Further, 1-1.5nmol/ul DSPE-PEG-Biotin solution is added into the autologous adipose-derived stem cell exosomes of the patient obtained through super-separation, so that the final concentration of the adipose-derived stem cell exosomes is 2.3-2.4 mug/ul, and then the exosomes are biotinylated after standing for 30-45min at 4 ℃.

Furthermore, the quantitative determination of the protein in the adipose-derived stem cell exosome is controlled to be 2-3 ug/ul.

Further, the medium containing the biotinylated exosome in the step (9) is a low-sugar DMEM medium containing 80-90ug/ml of the biotinylated exosome, 10-15% of Hyclone fetal bovine serum in volume fraction, and 1% of penicillin streptomycin mixed solution in volume fraction.

The photo-curable polycaprolactone is a commercially available material sold as EFL-PCLMA-3080 by Yongqin spring Equipment, Suzhou.

The invention utilizes the construction technology that the stem cell exosome is combined with the degradable biological scaffold and is implanted into the in-situ regenerated bone tissue, and has wider application prospect due to the advantages of small damage, less rejection and the like. After the bone defect repairing scaffold is implanted, the exogenous implant can be completely replaced by the self tissue along with the degradation of the biomaterial. The method not only avoids large-area donor area damage (autologous bone graft transplantation), but also avoids the risk caused by the implantation of non-degradable materials (metal implantation). The printed fine structure bone repair scaffold has biological activity, can continuously release cytokines and promote the generation of peripheral blood vessels and the growth of tissues, meanwhile, the bone scaffold adopts absorbable materials such as fat stem cells, and can be gradually absorbed by a human body, and the cytokines released in the period can promote the growth of the tissues and the blood vessels to complete the in-situ regeneration of the bone tissues.

Compared with the prior art, the invention has the following beneficial effects:

(1) the scaffold with bioactivity can continuously release cell factors and promote the regeneration of blood vessels and bone tissues of a body.

(2) The problem that bioactive substances cannot be uniformly distributed in the scaffold with a complex volume structure is solved by a mode of completely biotinylating the surface of the scaffold;

(3) by applying the photoetching 3D printing technology, the bone defect bracket of the patient is accurately constructed, and the personalized treatment of the patient can be achieved.

Drawings

FIG. 1 is a flow chart of a method of the present invention;

FIG. 2 is a model view of a bone repair scaffold of the present invention;

FIG. 3 is a schematic view of a printed bone repair scaffold;

FIG. 4 is a graph of Fourier transform infrared spectroscopy results after photo-curable polycaprolactone-modified grafting;

FIG. 5 is a test chart of the vesicle (adipose-derived stem cell exosome) related traits of adipose-derived stem cells;

FIG. 6 is a related electron micrograph of the modified material grafted biotinylated exosomes;

FIG. 7 is an experimental diagram of an animal;

FIGS. 8 and 9 are graphs showing the results of animal experiments;

Detailed Description

The invention will be further described with reference to specific embodiments and the accompanying drawings.

Example 1

A manufacturing method of a bone defect repairing support comprises the following steps:

(1) the patient photographs a bone defect CT before an operation, stores the bone defect CT in DICOM format, introduces surgical planning software (ProPlan 2.1, Materialise NV, Belgium), and reconstructs the bone defect CT to obtain a three-dimensional model of the defect. The software can then automatically generate the desired bone tissue length, diameter and surgical plan. Exporting the scheme to CAD software in an STL format;

(3) dissolving a photoinitiator TPO-L in a concentration of 1.8% w/v and a light absorbent beta carotene in a concentration of 0.09% w/v in photocuring Polycaprolactone (PCLMA) to obtain liquid ink for printing a bone scaffold;

(4) performing aseptic treatment on the bone scaffold printing liquid ink obtained in the step (3), adding a centrifuge tube, repeatedly blowing and uniformly mixing a liquid transfer gun, placing the mixture in a centrifuge at 3700r/min for centrifuging for 4 minutes to remove bubbles, and then heating the mixture to 37.5 ℃ in a water bath for later use;

(5) and (3) carrying out layered slicing treatment on the model obtained in the step (2), putting the printing raw materials obtained in the step (4) into a photocuring 3D printer together, carrying out aseptic printing, and printing layer by layer to obtain the solid bone defect repair support with the porous structure.

(6) And (3) carrying out surface modification treatment on the porous bone defect repair scaffold obtained in the step (5), firstly placing the porous bone defect repair scaffold in a mixed solution of 0.21mol/L potassium permanganate and 0.45mol/L hydrochloric acid, carrying out sterile treatment on the mixed solution in advance, and oscillating the mixed solution for 9 hours at 140r/min in a constant temperature shaking table at 46 ℃ to expose partial hydroxyl on the surface of the scaffold so as to conveniently modify the scaffold by using biotin.

(7) Biotinylation treatment is carried out on the porous bone repair scaffold with the surface exposed with hydroxyl groups obtained in the step (6), and 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine (EDC) is dissolved in 10ml of anhydrous dichloromethane at the concentration of 0.37% w/v, 4-Dimethylaminopyridine (DMAP) at the concentration of 0.005% w/v and Biotin (Biotin) at the concentration of 0.5% w/v to prepare biotinylation working solution of the bone repair scaffold.

(8) And (4) performing sterile treatment on the working solution obtained in the step (7), placing the bone repair scaffold obtained in the step (6) in the working solution, and shaking for 13 hours by using a constant-temperature shaking table at 48 ℃ at 140r/mim to obtain the bone repair scaffold with biotinylation on the surface.

(9) And (3) placing the biotinylated scaffold obtained in the step (8) in a culture medium containing biotinylated exosomes, adding 20ug/ml of streptavidin, and placing the mixture at 37.5 ℃ for 40min to tightly combine the biotinylated exosomes with the biotinylated scaffold by using a biotin-avidin system.

The parameters of the 3D printing in the step (5) are as follows: the layer height is 22 mu m, the light intensity is 17mW/cm2, the base layer number is 11, the base layer exposure time is 13s, the lamella exposure time is 9s, the stripping distance is 7mm, the stripping speed is 19mm/min, the stripping recovery speed is 110mm/min, and the light source wavelength is 405 nm.

The preparation method of the biotinylation exosome in the step (9) comprises the steps of taking 100ul of patient autologous adipose-derived stem cell exosome obtained through super-separation (the quantitation of exosome protein is 2.3ug/ul), adding 10ul of 1nmol/ul DSPE-PEG-Biotin solution, and standing for 30min at 4 ℃ to biotinylate the exosome.

The medium containing the biotinylated exosomes in the step (9) is a low-sugar DMEM medium containing 80ug/ml of the biotinylated exosomes, 10% by volume Hyclone fetal bovine serum and 1% by volume penicillin streptomycin mixed solution.

The protein, mRNA and the like contained in the adipose-derived stem cell exosome are beneficial to the osteogenic effect and the blood vessel growth of the bone defect part and accelerate the repair of the bone defect. The bone scaffold grafted with the biotinylated exosome is applied to a radius defect model (critical bone defect is 1.5cm) of a New Zealand white rabbit, and the result is observed after 1 month.

Example 2

A manufacturing method of a bone defect repairing bracket comprises the following steps:

(3) dissolving a photoinitiator TPO-L in a concentration of 1.5% w/v and a light absorber beta carotene in a concentration of 0.06% w/v in photocuring Polycaprolactone (PCLMA) to obtain liquid ink for printing a bone scaffold;

(4) performing aseptic treatment on the bone scaffold printed liquid ink obtained in the step (3), adding a centrifuge tube, repeatedly blowing and uniformly mixing a liquid transfer gun, placing the mixture in a centrifuge at 3500r/min for centrifuging for 3 minutes to remove bubbles, and then heating the mixture to 37 ℃ in a water bath for later use;

(6) And (5) carrying out surface modification treatment on the porous bone defect repairing scaffold obtained in the step (5), firstly placing the porous bone defect repairing scaffold into a mixed solution of 0.25mol/L potassium permanganate and 0.5mol/L hydrochloric acid, carrying out aseptic treatment on the mixed solution in advance, and oscillating the mixed solution for 8 hours at a constant temperature of 45 ℃ at a speed of 120r/min in a shaking table so as to expose partial hydroxyl on the surface of the scaffold and conveniently modify the hydroxyl by utilizing biotin.

(7) Biotinylation treatment is carried out on the porous bone repair scaffold with the surface exposed with hydroxyl groups obtained in the step (6), 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) is dissolved in 10ml of anhydrous dichloromethane at the concentration of 0.35% w/v, 4-Dimethylaminopyridine (DMAP) is dissolved at the concentration of 0.006% w/v, and Biotin (Biotin) is dissolved at the concentration of 0.4% w/v to prepare biotinylation working solution of the bone repair scaffold.

(8) And (4) performing sterile treatment on the working solution obtained in the step (7), placing the bone repair scaffold obtained in the step (6) in the working solution, and shaking for 12 hours by a constant-temperature shaking table at 47 ℃ at 120r/mim to obtain the bone repair scaffold with biotinylation on the surface.

(9) And (3) placing the biotinylation support obtained in the step (8) in a culture medium containing biotinylation exosomes, adding 22ug/ml of streptavidin, and placing for 37min at 37 ℃, so that the biotinylation exosomes can be tightly combined with the biotinylation support by using a biotin-avidin system.

The parameters of the 3D printing in the step (5) are as follows: the layer height is 20 mu m, the light intensity is 15mW/cm2, the base layer number is 10, the base layer exposure time is 10s, the lamella exposure time is 13s, the stripping distance is 8mm, the stripping speed is 20mm/min, the stripping recovery speed is 120mm/min, and the light source wavelength is 405 nm.

The preparation method of the biotinylated exosome in the step (9) comprises the steps of taking 100ul of autologous adipose-derived stem cell exosome (the exosome protein ration is 2.3ug/ul) of a patient obtained by super-separation, adding 10ul of DSPE-PEG-Biotin solution of 1.2nmol/ul, and standing for 30min at 4 ℃ to biotinylate the exosome.

The medium containing the biotinylated exosomes in the step (9) is a low-sugar DMEM medium containing 85ug/ml of the biotinylated exosomes, 12% by volume Hyclone fetal bovine serum and 1% by volume penicillin streptomycin mixed solution.

The bone scaffold grafted with the biotinylated exosome is applied to a rat femur defect model, and the result is observed after 1 month.

FIG. 4 is a diagram showing the result of Fourier transform infrared spectroscopy after modification and grafting of the photocurable polycaprolactone, wherein the reference numeral 1: PCLMA No. 2: the absorption peak of the PCLMA treated by the hydrochloric acid-potassium permanganate solution can be seen at 3000-3500, which proves that the surface hydroxyl group exposure label is 3: biotinylation of PCLMA can be seen, an absorption peak appears at 1500-1550, which proves that biotinylation is successful, and biotin is stably grafted on PCLMA.

The extraction process of the adipose-derived stem cell exosomes comprises the following steps: 2ml of PBS was added to the culture dish, and the cells were scraped off using a cell scraper. Blowing with 1ml pipette, transferring the cell suspension into a centrifugal tube, centrifuging at 1500rpm for 5min, and removing the supernatant; add 1ml PBS to the centrifuge tube, blow and resuspend. The extruder was assembled, PBS was pushed back and forth to check the sealing, and the cells were pushed 5 times each using a 1 μm filter screen with a pore size of 10 μm, 5 μm in sequence. A total of 27ml of 30% (7.5ml 60% iodixanol +7.5ml PBS) and 10% (2ml 60% iodixanol +10ml PBS) iodixanol isolate was prepared. Pouring 10% separating medium into the super-separation tube, injecting 30% separating medium into the bottom of the super-separation tube with a flat-mouth long needle, slowly moving the vesicle sample into the upper layer of the super-separation tube while keeping away from shaking to avoid fusion of the separating medium, and if the super-separation tube is not filled with PBS, using PBS. Ultracentrifugation is carried out for 2h at 100000g and 4 ℃, then a liquid transfer gun is used for sucking out the middle-layer vesicle, after balancing, ultracentrifugation is carried out for 2h at 100000g and 4 ℃ again, supernatant is discarded, 200 mu L PBS is used for resuspension, and then the lower-layer vesicle is sucked out.

Performing a staining experiment on the extracted adipose-derived stem cell exosomes (vesicles), and as shown in fig. 5, staining the vesicles with Dil dye, and observing the visible fine vesicles under a light microscope; shooting single vesicle by an electron microscope; the grain size analysis shows that the grain size of the engineered capsule is mostly 30-100 nm.

The biotinylated scaffold is grafted with biotinylated exosome by using biotin-avidin, ultrasonic cleaning is carried out for 5-10s, and then observation is carried out under an electron microscope, as shown in figure 6, dense vesicles are found, and the vesicles are indicated to be stably grafted to the surface of the material.

As shown in the animal experiment of figure 7, 2.5-3kg of New Zealand white rabbits, a critical bone defect model of 1.5cm of radius, a stent is implanted, and the radius defect model of the New Zealand white rabbits is subjected to 0.625 thin-layer CT scanning forelimb reconstruction result for 1 month, as shown in figure 8, the bone repair effect of the experimental group is obviously better than that of the blank control group; the hindlimb reconstruction result of the rat femoral defect model after 1 month 0.625 thin-layer CT scanning is shown in figure 9, and the bone repair effect of the experimental group is obviously superior to that of the blank control group.

The fine structure bone repair scaffold printed by the invention has biological activity, can continuously release cytokines and promote the generation of peripheral blood vessels and the growth of tissues, and meanwhile, the bone scaffold is made of absorbable materials such as PCLMA (polycaprolactone methacrylate-maleic anhydride) and can be gradually absorbed by a human body, and the cytokines released in the period can promote the growth of the tissues and the blood vessels to complete the in-situ regeneration of the bone tissues.

The above description is intended only to describe the embodiments of the present invention in detail, and the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made on the design concept of the present invention should be included in the protection scope of the present invention.

Claims

1. A preparation method of a bone defect repair bracket is characterized by comprising the following steps:

(1) obtaining the imaging data of the bone defect part of a patient in a clinical imaging CT and MRI scanning mode, and then carrying out three-dimensional modeling by utilizing CAD software to obtain a lamellar STL format file;

(2) designing an individualized bone repair support suitable for the bone defect part of the patient based on the data analysis and the model making;

(3) dissolving a photoinitiator TPO-L in a concentration of 1.0-2.0% w/v and a light absorbent beta-carotene in a concentration of 0.05-0.1% w/v in photocuring polycaprolactone to obtain liquid ink for printing a bone scaffold;

(4) performing aseptic treatment on the bone scaffold printing liquid ink obtained in the step (3), adding the bone scaffold printing liquid ink into a centrifuge tube, placing the centrifuge tube in a centrifuge for 3-5 minutes at 3000-4000r/min to remove bubbles, and heating the bone scaffold printing liquid ink to 37-37.5 ℃ in a water bath pan for later use;

(5) carrying out layered slicing treatment on the model obtained in the step (2), putting the printing raw materials obtained in the step (4) into a photocuring 3D printer together, carrying out aseptic printing, and printing layer by layer to obtain a solid bone defect repairing support with a porous structure;

(6) placing the porous bone defect repair scaffold obtained in the step (5) in a mixed solution containing 0.20-0.25mol/L potassium permanganate and 0.4-0.5mol/L hydrochloric acid, and oscillating for 8-9h at a constant temperature of 45-48 ℃ and a shaking table at a speed of 120-150 r/min;

(7) dissolving 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine at the concentration of 0.3% -0.4% w/v, 4-dimethylaminopyridine at the concentration of 0.005% -0.007% w/v and biotin at the concentration of 0.4% -0.5% w/v in anhydrous dichloromethane to prepare biotinylated working solution of the bone repair scaffold;

(8) performing aseptic treatment on the working solution obtained in the step (7), then placing the bone repair scaffold obtained in the step (6) in the working solution, and shaking the bone repair scaffold with a constant temperature shaking table at 47-48 ℃ for 12-14h at a speed of 120-;

(9) and (3) placing the biotinylated stent obtained in the step (8) in a culture medium containing biotinylated exosomes, adding streptavidin according to the ratio of 20-25ug/ml of the culture medium, placing for 30-45min at 37-37.5 ℃, and tightly combining the biotinylated exosomes on the biotinylated stent by using a biotin-avidin system to obtain the stent for promoting bone defect repair.

2. The method for preparing a bone defect repair scaffold according to claim 1, wherein: the parameters of the 3D printing in the step (5) are as follows: layer height 20-25 μm, lightStrong 15-20mW/cm²10-12 layers of base layer, 10-15s of base layer exposure time, 8-13s of lamella exposure time, 6-8mm of stripping distance, 18-20mm/min of stripping speed, 100 mm/min of stripping recovery speed and 405nm of light source wavelength.

3. The method for preparing a bone defect repair scaffold according to claim 1, wherein: and (3) filtering the mixed solution by using a 0.22um sterile filtration membrane for sterile treatment before adding the mixed solution into the step (6).

4. The method for preparing a bone defect repair scaffold according to claim 1, wherein: and (4) taking the exosomes in the biotinylated exosomes in the step (9) as autologous adipose-derived stem cell exosomes of the patient, obtaining adipose tissues of the patient by a suction method, digesting and extracting the adipose-derived stem cells by lipase, collecting supernatant when liquid is changed every time, centrifuging for 2 hours by 100000G in an ultracentrifuge, collecting bottom vesicles, and storing at-80 ℃ for later use.

5. The method for preparing a bone defect repair scaffold according to claim 4, wherein the biotinylated exosomes in the step (9) are prepared by the following steps: adding 1-1.5nmol/ul DSPE-PEG-Biotin solution into the super-separated autologous adipose-derived stem cell exosome of the patient to ensure that the final concentration of the adipose-derived stem cell exosome is 2.3-2.4 mu g/mu l, and then standing for 30-45min at 4 ℃ to biotinylate the exosome.

6. The method for preparing a bone defect repair scaffold according to claim 5, wherein the amount of protein in the adipose-derived stem cell exosomes is controlled to be 2-3 ug/ul.

7. The method for preparing a bone defect repair scaffold according to claim 1, wherein: the culture medium containing the biotinylated exosome in the step (9) is a low-sugar DMEM culture medium containing 80-90ug/ml of the biotinylated exosome, 10-15% of Hyclone fetal bovine serum in volume fraction and 1% of penicillin streptomycin mixed solution in volume fraction.