Exosome functionalized bone regeneration topological fibrous scaffold and preparation method thereof

Abstract

The application discloses an exosome functionalized bone regeneration topological fiber scaffold and a preparation method thereof. The preparation method comprises the following steps: (1) preparing a fiber scaffold substrate by electrospinning of bioactive molecules and high molecular compounds with a degradation rate gradient difference; (2) soaking the fiber scaffold substrate in an aqueous solution to obtain a topological fiber scaffold with a micro-sodium secondary pore structure; (3) modifying the topological fiber scaffold with the micro-sodium secondary pore structure to make the surface of the topological fiber scaffold positively charged, and then loading exosomes on the fiber scaffold to obtain the exosome functionalized bone regeneration topological fiber scaffold. The method provided by the application is simple and effective in combination of the physical characteristics of the topological fiber scaffold itself and the biological clue regulation of vascularized bone regeneration. The preparation and modification method of the topological fiber scaffold is stable, repeatable, simple to operate and easy to scale up.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fiber scaffold, in particular to an exosome functionalized bone regeneration topological fiber scaffold and a preparation method thereof. BACKGROUND

[0002] Trauma, infection and tumor resection can all cause bone defects. According to statistics, there are more than 3 million patients with bone defects in China every year. Bone defect treatment is one of the important problems that orthopedic clinicians have been trying to solve for a long time. At present, the vascularization and biological functional repair of bone defects still face severe challenges. Therefore, the development of vascularization-promoting and biological functional bone defect repair scaffolds can reduce the pain and social and economic burden of patients, and has important significance for promoting the construction of "healthy China" and improving the quality of life and health level of the people. So far, based on the exploration of the structure-property relationship of scaffold material components, a series of bone regeneration repair scaffolds have been designed and developed, which have shown great potential in promoting bone performance. However, the newly formed bone often leads to bone sclerosis or bone necrosis due to the lack of effective new blood vessel network, so there are still great challenges in achieving biological functional repair of bone defects.

[0003] The strategy of loading biochemical factors or cells to promote vascularization to construct vascularized bone regeneration scaffolds has been widely reported. Most of the bone tissue regeneration scaffolds designed and constructed at present directly regulate the behavior of cells related to vascular formation to promote vascular formation, and few regulate the "angiogenesis-osteogenesis-osteoclast" three-way coupling effect in the process of vascularized bone regeneration to achieve vascularized bone regeneration. Exosomes, as an important tool for intercellular communication, are called "lipid nanoparticles in nature", and have been proven to play an important role in promoting angiogenesis-osteogenesis. Macrophages have strong plasticity and are highly involved in the bone immune response and vascularized bone regeneration mediated by endothelial cells, osteoblasts and osteoclasts. Therefore, macrophage-derived exosomes, as messengers of intercellular communication, are likely to play an important role in regulating the "angiogenesis-osteogenesis-osteoclast" three-way coupling effect to mediate vascularized bone regeneration, but there are few related reports. SUMMARY

[0004] In order to solve the above-mentioned deficiencies existing in the prior art, the purpose of the present application is to provide an exosome functionalized bone regeneration topological fiber scaffold and a preparation method thereof, which can effectively improve the preparation of existing fiber scaffolds and the problems of bone tissue engineering scaffolds in realizing vascularized bone tissue regeneration.

[0005] The technical scheme for solving the above technical problems of the present application is as follows: a preparation method of an exosome functionalized bone regeneration topological fiber scaffold is provided, comprising the following steps:

[0006] (1) preparing a fiber scaffold substrate by electrospinning of bioactive molecules and high molecular compounds with different degradation rates;

[0007] (2) soaking the fiber scaffold substrate in an aqueous solution to obtain a topological fiber scaffold with a micro-sodium secondary pore structure;

[0008] (3) modifying the topological fiber scaffold with a micro-sodium secondary pore structure to make its surface positively charged, and then loading exosomes onto the fiber scaffold to obtain an exosome functionalized bone regeneration topological fiber scaffold.

[0009] The fiber scaffold substrate is soaked in PBS buffer or water, and the biologically active ingredients in the fiber scaffold substrate degrade quickly, while the high molecular compounds degrade slowly, forming a degradation gradient difference. The topological fiber scaffold with a micro-sodium secondary pore structure can be obtained by soaking in an aqueous solution, taking advantage of the degradation gradient difference between the high molecular compounds and the biologically active molecules. The micro-sodium secondary pore structure defined here refers to the pores between the fibers being micron-sized, and the pores on the fibers being nanometer-sized. The topological fiber scaffold with a micro-sodium secondary pore structure can promote cell adhesion, migration, proliferation and differentiation. When implanted in vivo, it can promote the adhesion of BMSCs (bone marrow mesenchymal stem cells) and macrophages, and further promote osteogenic differentiation and regulate bone immune response, ultimately mediating bone regeneration. The exosomes loaded on the topological fiber scaffold can be macrophage-derived exosomes, which can act as messengers for cell-to-cell communication, regulate the interaction between endothelial cells, bone marrow mesenchymal stem cells and immune cells, and play an important role in regulating vascularized bone regeneration mediated by the "angiogenesis-osteogenesis-osteoclast" ternary coupling effect. The topological fiber scaffold provided in the present application combines the physical properties (micro-sodium secondary pore structure) and biological properties (functional exosomes) of the scaffold itself to regulate cell behavior and mediate vascularized bone regeneration.

[0010] On the basis of the above technical solution, the present application can also be improved as follows:

[0011] Further, the specific process of step (1) is as follows: dispersing the biologically active molecules in an organic solvent, adding a high molecular compound, mixing uniformly to obtain a spinning solution, and preparing a fiber scaffold substrate by electrospinning.

[0012] Further, the biologically active molecules in step (1) are fish collagen, gelatin and derivatives thereof; the high molecular compound is a synthetic or natural high molecular compound, which can be polycaprolactone, poly(lactic-co-glycolic acid), polylactic acid, polyurethane, polyamide, chitosan, fish collagen, gelatin and derivatives thereof. The weight of the biologically active molecules accounts for 5-60% of the weight of the high molecular compound, preferably the weight of the biologically active molecules accounts for 10-30% of the weight of the high molecular compound.

[0013] The bioactive molecules and high molecular compounds are not limited to the above-mentioned compounds, as long as the bioactive components and the high molecular compounds have a certain degradation rate gradient in the aqueous solution, and can be electrospun into fibers, that is, the degradation rate gradient difference of the bioactive molecules and the high molecular compounds is beneficial to the formation of the topological fiber pore structure, and further adjusts the physicochemical properties and cell behavior of the topological fiber scaffold.

[0014] Further, the organic solvent in step (1) is trifluoroethanol, hexafluoroisopropanol, acetone, trifluoroacetic acid or N,N-dimethylformamide.

[0015] Further, in step (1), the electrospinning process parameters are set as follows: voltage 5-15kV, push injection speed 0.1-2mL / h, and receiving distance 5-25cm; preferably, the process parameters are as follows: voltage 6-10kV, push injection speed 0.2-0.6mL / h, and receiving distance 10-20cm.

[0016] Further, in step (1), the fiber scaffold substrate is a uniaxial fiber, a coaxial fiber with a core-shell structure or a multi-axial structure fiber.

[0017] Further, in step (2), the aqueous solution is PBS buffer or water, and the soaking time is 3-14 days, preferably 3-7 days.

[0018] The beneficial effects of the above further technical solutions are: the content of the bioactive components in the fiber scaffold substrate and the soaking time in the PBS buffer or water are very important, the higher the content of the bioactive components, the easier the formation of the pore structure, and the soaking time directly affects the formation of the topological fiber pore structure, the pore size and distribution, the micro-nano pore structure of the topological fiber scaffold plays an important role in adjusting the physicochemical properties, degradation performance, biocompatibility and cell behavior of the material system, and the corresponding pore structure and density can be selected according to the specific application environment.

[0019] Further, in step (3), the process of modifying the topological fiber scaffold is as follows: the topological fiber scaffold is soaked in NaOH solution, washed with deionized water after taking out, and then the surface of the topological fiber scaffold is positively charged through the EDC / NHS / PEI / MES reaction system, and finally washed with physiological saline.

[0020] Further, the concentration of the NaOH solution is 0.1-1 mol / L, and the soaking time is 3-10 min; the molar ratio of EDC to NHS is 3:1-1:1, and the molar ratio of PEI to EDC is 1:3-1:1; the concentration of MES is 0.1-0.5 M, the pH is 5-6.5, and the added volume is 10-20 mL; preferably, the concentration of the NaOH solution is 0.1-0.5 mol / L, and the soaking time is 5 min; the molar ratio of EDC to NHS is 2:1-1:1, and the molar ratio of PEI to EDC is 1:2-1:1; the concentration of MES is 0.1-0.3 M.

[0021] Further, the reaction time of the topological fiber scaffold in the EDC / NHS / PEI / MES reaction system is 0.5-2 h, and preferably the reaction time is 0.5-1 h.

[0022] The beneficial effects of the above further technical solutions are: the fiber scaffold is soaked in a NaOH solution, the purpose of this treatment is to improve the hydrophilicity of the fiber scaffold, and carboxyl groups are generated on the fiber surface through hydrolysis, and then the scaffold is thoroughly cleaned with deionized water. After cleaning, the fiber scaffold is subjected to an EDC / NHS / PEI / MES reaction system, so that PEI is coupled to the fiber scaffold, and the surface of the fiber scaffold is positively charged. MES is a buffer in EDC / NHS / PEI / MES, and the molar ratio of EDC / NHS / PEI plays a more important role in the surface modification of the topological fiber scaffold. On the basis of the above specific molar ratio, the surface charge of the topological fiber scaffold can be effectively converted to a positive charge, which is beneficial for loading exosomes.

[0023] Further, the process of loading exosomes onto the fiber scaffold in step (3) is: placing the fiber scaffold with a positively charged surface in an aqueous solution containing exosomes, and after standing at 0-4℃ for 3-24 h, the exosome functionalized bone regeneration topological fiber scaffold is obtained.

[0024] Further, the concentration of the aqueous solution containing exosomes is 1×10 9 -1×10 15 / ml, and preferably the concentration is 1×10 11 -1×10 13 / ml; the standing time is preferably 4-12 h.

[0025] The beneficial effects of the above further technical solutions are: PEI has abundant amino groups, which can effectively load exosomes onto the surface of the topological fiber scaffold material through positive and negative charge interaction.

[0026] Further, the exosomes are M2 macrophage-derived exosomes, or M1 macrophage or bone marrow mesenchymal stem cell or bone marrow cell-derived exosomes.

[0027] Exosomes, as the messenger of cell-cell communication, can regulate the interaction of endothelial cells, bone marrow mesenchymal stem cells and immune cells, and can regulate the immune cell responsiveness around the implanted material, and plays an important role in regulating the vascularized bone regeneration mediated by the“angiogenesis-osteogenesis-osteoclastogenesis”ternary coupling effect.

[0028] The present application has the following beneficial effects:

[0029] The preparation and modification method of the topological fiber scaffold provided by the present application is simple and effective, the biologically active molecules and high molecular compounds with different degradation rate gradients are subjected to electrospinning and simple soaking treatment in the later stage, and the topological fiber scaffold with micro-nano secondary pore structure is prepared, the surface electric property of the topological fiber scaffold is converted into positive charge through the NaOH / EDC / NHS / PEI / MES reaction system, and the negatively charged exosomes can be loaded on the surface of the high molecular based topological fiber scaffold material through the charge effect.

[0030] The hydrophilicity of the topological fiber scaffold is significantly improved after surface modification, which is beneficial to cell adhesion, migration, proliferation and differentiation, and further promotes bone tissue regeneration.

[0031] The micro-nano secondary pore structure of the topological fiber scaffold improves the nanoscale roughness of the fiber scaffold, which is beneficial to cell adhesion and migration.

[0032] In addition, after the topological fiber scaffold is loaded with exosomes, not only the nanoscale roughness of the scaffold is further improved, but also the immune cell responsiveness after implantation of the scaffold is adjusted, the behaviors of endothelial cells, osteoblasts and osteoclasts are adjusted, and then the vascularized bone tissue regeneration is effectively realized.

[0033] The method for regulating vascularized bone regeneration by combining the physical properties of the topological fiber scaffold itself and biological clues provided by the present application is simple and effective, the preparation and modification method of the topological fiber scaffold is stable, repeatable, simple to operate and easy to scale up. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The topological fiber scaffold with different fish collagen contents after soaking in PBS buffer solution.

[0035] Figure 2 The XPS spectra of the topological fiber scaffold before and after modification.

[0036] Figure 3 The surface electric property results of the topological fiber scaffold before and after surface modification.

[0037] Figure 4 The structure, particle size, electric property and marker detection results of the exosomes.

[0038] Figure 5Scanning electron microscope images of topological fiber scaffold after soaking treatment and loading of exosomes.

[0039] Figure 6 Water contact angle of topological fiber scaffold.

[0040] Figure 7 Graph of focal adhesion expression level of BMSCs on different topological fiber scaffolds.

[0041] Figure 8 Graph of guided bone regeneration effect of exosome functionalized topological fiber scaffold. DETAILED DESCRIPTION

[0042] The following examples are intended to illustrate the present application and are not intended to limit the scope of the present application. Unless otherwise indicated, the conditions in the examples are conventional conditions or those recommended by the manufacturer. Unless otherwise indicated, the reagents or instruments used are conventional products available on the market.

[0043] Example 1

[0044] An exosome functionalized bone regeneration topological fiber scaffold, a preparation method thereof includes the following steps:

[0045] Step 1: 0.06 g of fish collagen was weighed and dispersed in 1 mL of hexafluoroisopropanol to obtain a dispersion liquid; 0.2 g of polylactic acid-glycolic acid was weighed and added to the obtained dispersion liquid, and a constant temperature shaker was used to shake and mix for 60 min to obtain a spinning liquid A; 0.12 g of polycaprolactone was weighed and added to the obtained dispersion liquid, and a constant temperature shaker was used to shake and mix for 45 min to obtain a spinning liquid B;

[0046] Step 2: The spinning liquids A and B obtained in step 1 were respectively loaded into 5 mL syringes and placed in two push injection devices of an electrospinning machine, the spinning liquids A and B were respectively connected with the outer layer and the inner layer of the coaxial needle, and a fish collagen / polylactic acid-glycolic acid / polycaprolactone composite fiber scaffold (abbreviated as Untreated PP) was prepared by electrospinning, and the obtained fiber scaffold was placed in a vacuum drying box for drying for one week; wherein the electrospinning voltage was 8 kV, the push injection speed of the spinning liquids A and B was 0.4 ml / h and 0.2 mL / h respectively, the receiving distance was 15 cm, and a flat plate receiver was used for receiving;

[0047] Step 3: The fiber scaffold obtained in step 2 was immersed in PBS buffer and placed in a constant temperature shaker at a rotation speed of 120 r / min for 3 days, and the topological fiber scaffold (abbreviated as PP) with micro-nano pore structure obtained after soaking treatment was placed in a vacuum drying box for drying for one week;

[0048] Step 4: The PP fiber scaffold obtained in step 3 was immersed in 0.2 mol / L NaOH solution for 5 min, and then repeatedly washed with deionized water for three times.

[0049] Step 5: The fiber scaffold obtained in step 4 was placed in a 20 mL reaction solution containing 2.45 g PEI, 0.0446 g EDC and 0.0168 g NHS with pH = 5.5 MES, and after 0.5 hours of reaction, it was gently washed twice with deionized water to obtain a PEI surface modified topological fiber scaffold;

[0050] Step 6: The exosomes were extracted from RAW264.7 (mouse macrophage) cell culture solution by ultracentrifugation (the following centrifugation operations were sequentially performed: 300 g centrifugation for 10 min, 3000 g centrifugation for 10 min, 10000 g, 4°C centrifugation for 30 min, 110000 g, 4°C centrifugation for 70 min, 110000 g, 4°C centrifugation for 70 min), and the modified topological fiber scaffold obtained in step 5 was placed in an exosome solution with a concentration of 1×10 12 Individuals / ml, and after being placed at 4°C for 12 hours, it was taken out and gently washed twice with deionized water to prepare an exosome functionalized topological fiber scaffold.

[0051] Example 2:

[0052] An exosome functionalized bone regeneration topological fiber scaffold, and a preparation method thereof, the preparation method comprising the following steps:

[0053] Step 1: 0.04 g of fish collagen was weighed and dispersed in 1 mL of hexafluoroisopropanol to obtain a dispersion liquid; 0.2 g of polylactic acid-glycolic acid was weighed and added to the obtained dispersion liquid, and a constant temperature shaker was used for shaking and uniformity for 60 min to obtain a spinning liquid A; 0.12 g of polycaprolactone was weighed and added to the obtained dispersion liquid, and a constant temperature shaker was used for shaking and uniformity for 45 min to obtain a spinning liquid B;

[0054] Step 2: The spinning liquids A and B obtained in step 1 were respectively loaded into 5 mL syringes and placed in two push injection devices of an electrospinning machine, and the spinning liquids A and B were respectively connected with the outer layer and the inner layer of the coaxial needle, and a fish collagen / polylactic acid-glycolic acid / polycaprolactone composite fiber scaffold (abbreviated as Untreated PP) was prepared by electrospinning, and the obtained fiber scaffold was placed in a vacuum drying box for drying for one week; wherein the electrospinning voltage was 8 kV, the push injection speed of the spinning liquids A and B was 0.4 ml / h and 0.2 mL / h respectively, the receiving distance was 15 cm, and a flat plate receiver was used for receiving;

[0055] Step 3: The fiber scaffold obtained in step 2 was immersed in a PBS buffer solution and placed in a constant temperature shaker for oscillation at a speed of 120 r / min for 5 days, and the topological fiber scaffold (abbreviated as PP) with micro-nano pore structure obtained after soaking treatment was placed in a vacuum drying box for drying for one week;

[0056] Step 4: The PP fiber scaffold obtained in step 3 was immersed in a 0.2 mol / L NaOH solution for 5 min and then rinsed with deionized water three times;

[0057] Step 5: The fiber scaffold obtained in step 4 was placed in a 20 mL pH 5.5 MES reaction solution containing 2.45 g PEI, 0.0446 g EDC and 0.0168 g NHS, and reacted for 0.5 h. After that, the fiber scaffold was rinsed with deionized water twice to obtain a PEI surface-modified topological fiber scaffold;

[0058] Step 6: Exosomes were extracted from RAW264.7 cell culture solution by ultracentrifugation (the following centrifugation operations were sequentially performed: 300 g centrifugation for 10 min, 3000 g centrifugation for 10 min, 10000 g centrifugation at 4°C for 30 min, 110000 g centrifugation at 4°C for 70 min, and 110000 g centrifugation at 4°C for 70 min). The modified topological fiber scaffold obtained in step 5 was placed in an exosome solution with a concentration of 1×10 12 9 / ml, and stored at 4°C for 12 h. After that, the fiber scaffold was rinsed with deionized water twice to obtain an exosome functionalized topological fiber scaffold.

[0059] Example 3:

[0060] An exosome functionalized bone regeneration topological fiber scaffold, and a preparation method thereof, are provided.

[0061] Step 1: 0.036 g of fish collagen was weighed and dispersed in 1 mL of trifluoroethanol to obtain a dispersion liquid. 0.12 g of polycaprolactone was weighed and added to the obtained dispersion liquid, and the mixture was shaken uniformly for 60 min by using a constant temperature shaker to obtain a spinning solution;

[0062] Step 2: The spinning solution obtained in step 1 was loaded into a 5 mL syringe and placed in a push injection device of an electrospinning machine. A fish collagen / polycaprolactone composite fiber scaffold (abbreviated as Untreated PCL) was prepared by electrospinning. The obtained fiber scaffold was dried in a vacuum drying box for one week. In the process, the electrospinning voltage was 8 kV, the push injection speed was 0.4 ml / h, the receiving distance was 15 cm, and a flat plate receiver was used.

[0063] Step 3: The fiber scaffold obtained in step 2 was immersed in a PBS buffer and placed in a constant temperature shaker for oscillation at a speed of 120 r / min for 5 days. The topological fiber scaffold (abbreviated as PCL) with micro-nano pore structure obtained after immersion treatment was dried in a vacuum drying box for one week.

[0064] Step 4: The PCL fiber scaffold obtained in step 3 was immersed in a 0.2 mol / L NaOH solution for 5 min and then rinsed with deionized water three times;

[0065] Step 5: The fiber scaffold obtained in step 4 was placed in a 20 mL reaction solution containing 2.45 g PEI, 0.0446 g EDC and 0.0168 g NHS, and the pH was 5.5. After 0.5 hours of reaction, the scaffold was gently washed twice with deionized water to obtain a PEI surface modified topological fiber scaffold;

[0066] Step 6: Exosomes were extracted from RAW264.7 cell culture solution by ultracentrifugation (the following centrifugation operations were sequentially performed: 300 g centrifugation for 10 min, 3000 g centrifugation for 10 min, 10000 g centrifugation at 4°C for 30 min, 110000 g centrifugation at 4°C for 70 min, and 110000 g centrifugation at 4°C for 70 min). The modified topological fiber scaffold obtained in step 5 was placed in an exosome solution with a concentration of 1×10 13 individuals / ml, and after 4 hours of storage at 4°C, the scaffold was taken out and gently washed twice with deionized water to obtain an exosome functionalized topological fiber scaffold.

[0067] Experimental Example 1: Detection of micro-nano secondary pore structure of topological fiber scaffold

[0068] Taking Example 2 as an example, the morphology of the topological fiber scaffold with different fish collagen contents after immersion in PBS buffer was observed by scanning electron microscopy, and the results are shown in Figure 1 The topological fiber scaffold showed a typical secondary pore structure, with nanoscale pores on the fibers and micrometer-scale pores between the fibers, and the number and size of the pores increased significantly with increasing fish collagen content.

[0069] Experimental Example 2: Surface chemical detection of topological fiber scaffold before and after modification

[0070] Taking Example 2 as an example, the surface element chemical state of the topological fiber scaffold was detected by X-ray photoelectron spectroscopy, and the results are shown in Figure 2 which showed that the topological fiber scaffold was successfully surface modified by PEI.

[0071] Experimental Example 3: Surface electrical property detection of topological fiber scaffold

[0072] The topological fiber scaffold was prepared by electrospinning, and the scaffold was surface modified by a NaOH / EDC / NHS / PEI / MES reaction system. The surface electrical properties of the fiber scaffold before and after modification were further detected by solid Zeta potential, and the results are shown in Figure 3 which showed that the topological fiber scaffold before modification was negatively charged, and the topological fiber scaffold after modification was positively charged.

[0073] Experimental Example 4: Exosome identification

[0074] Exemplified by Example 1, the structure of the extracted exosomes was observed by transmission electron microscopy, the particle size and electrical properties of the exosomes were detected by a nanoparticle size tracking analyzer, and the expression of exosome markers was detected by immunoblotting, and the results are shown in Figure 4 Exosomes have a typical double-layer structure, with a size of about 119 nm and a negative surface charge.

[0075] Experimental Example 5: Topological fiber scaffold morphology observation

[0076] The morphology of the topological fiber scaffold was observed by scanning electron microscopy after treatment with PBS buffer and loading of exosomes, and the results are shown in Figure 5 After immersion treatment with PBS buffer, a large number of pore structures appeared on the fibers, showing obvious micro-nano secondary pore structures, and after further loading of exosomes, exosomes were clearly visible attached to the fibers on the topological fiber scaffold.

[0077] Experimental Example 6: Detection of the hydrophilic properties of the topological fiber scaffold

[0078] The hydrophilic properties of the fiber scaffold were detected by a contact angle measuring instrument, and the results are shown in Figure 6 The hydrophilic properties of the PP scaffold were improved after immersion treatment, and further improved after loading of exosomes, which is conducive to cell adhesion, migration, and proliferation.

[0079] Experimental Example 7: Detection of the ability of the topological fiber scaffold to promote cell adhesion

[0080] BMSC cells were seeded onto the three fiber scaffolds, and after 3 days of culture, the cell morphology and focal adhesion expression levels were detected by immunofluorescence staining, and the results are shown in Figure 7 The micro-nano secondary pore structure and exosomes are both conducive to BMSC adhesion.

[0081] Experimental Example 8: Detection of bone tissue regeneration performance

[0082] The topological fiber scaffold PP and the exosome-functionalized topological fiber scaffold PP-Exo were implanted into a rat skull critical defect model, and after 10 weeks, the skull repair effect was observed, and the Micro-CT reconstruction results are shown in Figure 8 A large number of new bone formation was observed in the defect area of the PP-Exo group, almost completely covering the defect area, indicating that the prepared exosome-functionalized topological fiber scaffold has good guided bone tissue regeneration effect and has a broad application prospect in the field of bone tissue engineering.

[0083] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing an exosome-functionalized bone regeneration topological fiber scaffold, characterized in that, Includes the following steps: (1) A spinning solution is prepared by mixing bioactive molecules and polymers with different degradation rate gradients, and then a fiber scaffold substrate with micron-sized pores is prepared by electrospinning. (2) The fiber scaffold substrate is immersed in an aqueous solution to degrade it, forming nanopores on the fiber to obtain a topological fiber scaffold with a micro-sodium secondary pore structure; wherein, the micro-sodium secondary pore structure is that the pores between the fibers are micrometer-scale and the pores on the fibers are nanometer-scale; the aqueous solution is PBS buffer or water; (3) Modify the topological fiber scaffold with microsodium secondary pore structure to make its surface positively charged, and then load exosomes onto the fiber scaffold to obtain an exosome-functionalized bone regeneration topological fiber scaffold.

2. The method for preparing the exosome-functionalized bone regeneration topological fiber scaffold according to claim 1, characterized in that, Step (1) involves dispersing bioactive molecules in an organic solvent, adding a polymer compound, mixing well, obtaining a spinning solution, and preparing a fiber scaffold substrate by electrospinning.

3. The method for preparing the exosome-functionalized bone regeneration topological fiber scaffold according to claim 1 or 2, characterized in that, In step (1), the bioactive molecules are fish collagen, gelatin and their derivatives; the polymers are polycaprolactone, polylactic acid-glycolic acid, polylactic acid, polyurethane, polyamide, chitosan, fish collagen, gelatin and their derivatives; the bioactive molecules account for 5-60% of the weight of the polymers.

4. The method for preparing the exosome-functionalized bone regeneration topof-fiber scaffold according to claim 1 or 2, characterized in that, In step (1), the electrospinning process parameters are set as follows: voltage 5-15kV, injection speed 0.1-2mL / h, and receiving distance 5-25cm.

5. The method for preparing the exosome-functionalized bone regeneration topof-fiber scaffold according to claim 1, characterized in that, The soaking time in step (2) is 3-14 days.

6. The method for preparing the exosome-functionalized bone regeneration topof-fiber scaffold according to claim 1, characterized in that, The modification process of the topological fiber scaffold in step (3) is as follows: the topological fiber scaffold is immersed in NaOH solution, taken out and washed with deionized water, then the surface of the topological fiber scaffold is positively charged by the EDC / NHS / PEI / MES reaction system, and finally washed with physiological saline.

7. The method for preparing the exosome-functionalized bone regeneration topof-fiber scaffold according to claim 6, characterized in that, The concentration of NaOH solution is 0.1-1 mol / L, and the soaking time is 3-10 min; the molar ratio of EDC to NHS is 3:1-1:1, and the molar ratio of PEI to EDC is 1:3-1:1; the concentration of MES is 0.1-0.5 M, the pH is 5-6.5, and the addition volume is 10-20 mL; the reaction time of the topological fiber scaffold in the EDC / NHS / PEI / MES reaction system is 0.5-2 h.

8. The method for preparing the exosome-functionalized bone regeneration topof-fiber scaffold according to claim 1, characterized in that, The process of loading exosomes onto the fiber scaffold in step (3) is as follows: the fiber scaffold with positive charge on its surface is placed in an aqueous solution containing exosomes, and after standing at 0-4℃ for 3-24 hours, it is taken out to obtain an exosome-functionalized bone regeneration topological fiber scaffold.

9. The method for preparing the exosome-functionalized bone regeneration topof-fiber scaffold according to claim 8, characterized in that, The concentration of the aqueous solution containing exosomes is 1×10 9 -1×10 15 The sample size is 1 / ml, and the standing time is 4-12 hours.

10. An exosome-functionalized bone regeneration topological fiber scaffold prepared by the preparation method according to any one of claims 1-9.

Images