CN117339009A

CN117339009A - DFO@GMs-pDA/PN composite stent for promoting vascularization and bone formation as well as preparation method and application thereof

Info

Publication number: CN117339009A
Application number: CN202311351856.9A
Authority: CN
Inventors: 赵立星; 汪饶开卷; 陈柔臻; 周洪玲
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2023-10-19
Filing date: 2023-10-19
Publication date: 2024-01-05

Abstract

The invention belongs to the technical fields of biomedical materials and biomedical engineering, and discloses a DFO@GMs-pDA/PN composite scaffold for promoting vascularization and bone formation, and a preparation method and application thereof. According to the invention, by utilizing the electrostatic spinning, pDA modification and emulsification-extraction technology, DFO is wrapped in GMs and loaded in a PN electrospun membrane modified by the pDA surface, so that the DFO@GMs-pDA/PN composite scaffold capable of promoting vascularized bone regeneration is constructed. Wherein, the surface DFO@GMs begins to disintegrate and release DFO after implantation and reaches the maximum drug release rate after 48 hours, thereby promoting the growth of new blood vessels, the recruitment of stem cells and the secretion and presentation of related factors in early stage of bone regeneration and providing an important blood supply basis for bone regeneration.

Description

DFO@GMs-pDA/PN composite stent for promoting vascularization and bone formation as well as preparation method and application thereof

Technical Field

The invention relates to the technical fields of biomedical materials and biomedical engineering, in particular to a DFO@GMs-pDA/PN composite stent for promoting vascularization and bone formation, and a preparation method and application thereof.

Background

Bone tissue is a composite matrix composed of mineral phases that provide mechanical strength, osteoconductive matrix, and collagen phases that play a central role in progenitor cell differentiation, mineralization and maturation. Meanwhile, bone is a highly vascularized tissue that relies on the connection between blood vessels and bone cells to maintain its integrity. Bone regeneration is a complex, diverse and coordinated process, wherein the key to bone tissue regeneration is the coupling of the angiogenies to the bone formation. The vascular network is not only used as a way for recruiting bone progenitor cells and endothelial progenitor cells for bone tissues, but also is a channel for ensuring sufficient oxygen and nutrient substances of the bone tissues and timely discharging metabolic wastes, and simultaneously participates in the regulation and control of various cells and signal molecules in the bone regeneration process, so that the internal balance of the bone tissues can be maintained. Vascularized bone regeneration is a key to ensuring complete bone healing, accelerating the bone repair process, improving bone remodeling quality, while achieving osteogenic-angiogenic coupling in the bone healing process is a core problem that is facing in current bone tissue engineering (Bone tissue engineering, BTE) techniques.

In recent years, the scaffold designed in bone tissue regeneration research mainly adopts technologies such as electrostatic spinning, hydrogel, freeze drying and 3D printing, wherein the electrostatic spinning is a technology for preparing fine fibers from a polymer solution by utilizing electrostatic force, and has the advantages of extremely high surface area volume ratio, adjustable porosity, extensibility capable of adapting to various sizes and shapes, capability of controlling the composition of nanofibers and the like. The following advantages exist when electrospinning technology is used in BTE: (1) Its structure mimics bone tissue extracellular matrix (ECM); (2) The porous ceramic material has high porosity and penetrability, is convenient for diffusion of nutrient substances and is beneficial to growth, proliferation and migration of cells; (3) Has higher entrapment efficiency on bioactive factors, polypeptides or medicaments. PCL is approved by FDA for biomedicine, has proper tensile property, biocompatibility, osseointegration and biodegradability, has proper degradation speed, can not degrade too fast to support tissue regeneration, and can not degrade too slow to influence tissue regeneration, and is suitable as a BTE scaffold material. However, PCL itself presents a strong hydrophobicity that tends to prevent cell migration and delay integration of the scaffold material with the host tissue. Thus, pure PCL scaffolds suffer from poor cell adhesion and lack of bioactivity, which makes them problematic in that the scaffold-cell interface lacks biological function. NCs is a two-dimensional nano bone tissue engineering material, and has multiple functions, such as an inherent osteoinductive effect, and can improve mechanical properties, drug release capacity and the like. Loading NCs into polymer nanofibers can enhance and improve the mechanical properties and osteogenic activity of the scaffold. Secondly, the addition of NCs can improve the hydrophobicity of PCL, promote the adsorption of water, hydrolyze and degrade PCL chains, and is more beneficial to cell attachment, proliferation and migration. At the same time, NCs can increase the roughness of electrospun fibers and have been shown to be potentially osteoinductive in itself and promote biomineralization. Furthermore, NCs also have higher specific surface area and charge anisotropy, and exhibit strong drug binding and sustained release ability to various molecules.

Microspheres are spherical microparticles that allow drug molecules to be dispersed therein, encapsulating and carrying the drug with its very tiny scaffold. Gelatin is a collagen degradation product in animal connective tissue or epidermal tissue, is a linear polymer formed by crosslinking 18 amino acids and polypeptides, has low antigenicity, good degradability and biocompatibility, and is also rich in arginine-glycine-aspartic acid sequences which have promotion effects on cell adhesion and migration.

DFO promotes vascularization coupled osteogenesis mainly through several pathways: (1) In the early stages of bone regeneration, DFO first promotes the expression of a range of angiogenic signals and active factors by activating HIF-1 alpha signaling pathways to promote neovascularization in the tissue of the target area. These new blood vessels serve as necessary channels for establishing communication with adjacent tissues in the defect area, and provide nutrition and oxygen, transport waste, etc. to seed cells while recruiting osteoprogenitor cells or osteogenic precursor cells, etc. for tissue regeneration to support the growth, migration, and differentiation of stem cells. (2) DFO has been shown to promote BMP-2 secretion by endothelial cells to promote osteogenic differentiation of mesenchymal stem cells; mesenchymal stem cells, in turn, can secrete VEGF to promote vascularization under this stimulus, with the interaction of the two cells effecting a vascular-osteogenic coupling. (3) DFO can inhibit differentiation of osteoclast precursor cells in remodelled/remodelled bone tissue, indirectly promoting osteogenesis by preventing bone loss. Past researches show that the release time of DFO after being physically adsorbed on the surface of the stent material is about 8 hours, and the effects of fixed-point application and controlled slow release cannot be achieved.

The angiopoiesis/osteogenesis active factors carried or added by the traditional BTE scaffold have limitations due to difficult preparation, easy inactivation, inability to target application, side effects and the like.

Disclosure of Invention

The invention aims to provide a DFO@GMs-pDA/PN composite stent for promoting vascularization and bone formation, and a preparation method and application thereof, so as to solve the problems that the vascularization/bone formation active factors carried or added by the traditional BTE stent are difficult to prepare, easy to inactivate, incapable of being applied in a targeting manner, have side effects and the like.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the invention provides a preparation method of a DFO@GMs-pDA/PN composite stent for promoting vascularization and bone formation, which comprises the following steps:

(1) Preparation of PN stent main body: mixing the nanoclay, polycaprolactone and an electrospinning solvent to obtain a mixed solution; carrying out electrostatic spinning on the mixed solution to obtain a PN bracket main body;

(2) Preparation of polydopamine modified PN stent main body: mixing dopamine powder and a tris buffer solution to obtain a mixed solution; immersing the PN stent main body into the mixed solution for reaction to obtain a polydopamine modified PN stent main body;

(3) Preparation of dfo@gms: mixing gelatin, deferoxamine and water to obtain solution A; mixing mineral oil and Span-80 to obtain solution B; carrying out cross-linking reaction on the solution A, the solution B and glutaraldehyde to obtain DFO@GMs;

(4) Preparation of DFO@GMs-pDA/PN composite scaffold: mixing DFO@GMs, ethanol and phosphate buffer salt solution to obtain a mixed solution; and immersing the polydopamine modified PN stent main body into the mixed solution for reaction to obtain the DFO@GMs-pDA/PN composite stent.

Preferably, in the step (1), the electrospinning solution comprises N, N-dimethylformamide and dichloromethane; the mass ratio of the N, N-dimethylformamide to the dichloromethane is 3-4: 1, a step of; the mass ratio of the nanoclay to the polycaprolactone to the electrospinning solvent is 0.12-0.15: 1.5 to 2.5: 8-10.

Preferably, in the step (1), the temperature of the mixing is 55 to 65 ℃, the stirring rate of the mixing is 100 to 200r/min, and the mixing time is 50 to 60min.

Preferably, in the step (1), parameters of the electrospinning are set as follows: the positive voltage is 11.5-12.5 kV, the negative voltage is 2.3-2.6 kV, the injection speed is 0.14-0.16 mm/min, the receiving distance is 8-12 cm, the receiving speed is 30-35 r/min, and the receiving time is 2.5-3 h.

Preferably, in the step (2), the mass ratio of the dopamine powder to the tris in the tris buffer is 0.2:0.11 to 0.12; the reaction time is 10-14 h, and the stirring rate of the reaction is 200-250 r/min.

Preferably, in the step (3), the mass-volume ratio of the gelatin, the deferoxamine and the water is 2-3 g:0.01 to 0.02g: 20-25 mL; the volume ratio of the mineral oil to Span-80 is 45-50: 1, a step of; the volume ratio of Span-80 to glutaraldehyde is 2:0.03 to 0.05; the volume fraction of glutaraldehyde is 45-50%.

Preferably, in the step (3), the temperature of the crosslinking reaction is 3 to 4 ℃, and the time of the crosslinking reaction is 20 to 30 minutes.

Preferably, in the step (4), the mass-volume ratio of dfo@gms, ethanol and phosphate buffer salt solution is 200mg: 47-50 mL: 45-51 mL; the reaction time is 10-14 h, and the stirring rate of the reaction is 200-250 r/min.

The invention also provides the DFO@GMs-pDA/PN composite scaffold prepared by the preparation method of the DFO@GMs-pDA/PN composite scaffold for promoting vascularization and bone formation.

The invention also provides application of the DFO@GMs-pDA/PN composite scaffold in scaffolds used for bone tissue engineering.

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

(1) DFO as a small molecule drug used in the present invention can promote complex angiogenic processes of multifactorial, multipass and co-coordination of multiple active factors by inhibiting prolyl hydroxylase domain Proteins (PHDs) from activating hypoxia inducible factor-1α (Hypoxiainducible factor-1α, HIF-1α) signaling pathways. Compared with the limitation of transmitting only 1-2 growth factors in the prior study, the DFO can be coupled with the bone after promoting angiogenesis, and can promote the healing of the bone to the greatest extent in the tissue regeneration process.

(2) The invention utilizes electrostatic spinning and emulsification-extraction technology, takes PN electrospun membrane as the main body of the composite scaffold, combines drug-loaded microsphere DFO@GMs with the PN electrospun scaffold in a pDA surface modification mode, and prepares the composite scaffold DFO@GMs-pDA/PN which can locally slowly release angioplastic and osteogenic factors, and the two cooperate to enable the angiogenesis and the osteogenesis to be coupled so as to promote the regeneration of vascularized bones. Wherein, the DFO@GMs on the outermost surface starts to disintegrate and release DFO after implantation and reaches the maximum drug release rate after 48 hours, thereby promoting the growth of new blood vessels, the recruitment of stem cells and the secretion and presentation of related factors in the early stage of bone regeneration and providing an important blood supply basis for bone regeneration; the PN electrospun scaffold with performance matched with the bone structure of the defect model can be stably present in the defect area in the bone regeneration process, simulate the extracellular matrix of bone tissue, provide space support for proliferation and differentiation of stem cells and new tissue, promote osteogenesis differentiation and subsequent mineralization, and realize effective promotion of bone regeneration.

(3) The composite scaffold after pDA surface modification and DFO@GMs loading has lower cytotoxicity, can be used for normal adhesion, growth and proliferation of cells to a certain extent, and the cell compatibility of the composite scaffold indicates that the scaffold material has the basic characteristics of being used as a tissue engineering scaffold. In addition, the release of DFO from the microspheres in DFO@GM endows the DFO@GMs-pDA/PN composite scaffold with the capability of promoting endothelial cell angiogenesis through the stimulation and angiogenesis-related signaling of human umbilical vein endothelial cells (Humanumbilicalvein endothelial cells, HUVECs); meanwhile, the NCs loaded in the DFO@GMs-pDA/PN composite scaffold act on rat bone marrow mesenchymal stem cells (Rat bone marrow mesenchymal stem cells, rBMSCs) in cooperation with DFO, so that the osteogenic differentiation of rBMSCs can be effectively promoted, and the potential of promoting bone tissue regeneration is provided.

(4) In vivo studies were performed by constructing a rat skull defect repair model and implanting a dfo@gms-pDA/PN composite scaffold. The result shows that the implanted DFO@GMs-pDA/PN composite stent can stably exist in a bone defect area, has good in vivo biocompatibility, is beneficial to healing of damaged periosteum and recovery of continuity, can maintain an osteogenic space and support regeneration of bone tissues in the defect area, and obviously promotes repair of rat skull defects; wherein the new bone tissue has the same structure as the original bone tissue and has mature lamellar bone, can maintain brain space and provide the functions of protecting soft tissue and supporting skull morphology. The DFO@GMs-pDA/PN composite scaffold obtained by the invention can effectively promote vascularized bone regeneration of skull and accelerate repair of bone defect through the vascularization coupling bone formation process in a rat body, is a potential BTE scaffold, and provides a new direction for bone regeneration research and clinical treatment of bone defect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of a preparation flow of a DFO@GMs-pDA/PN composite stent;

FIG. 2 is an SEM image of polycaprolactone, PN electrospun film obtained in step (1), and pDA/PN obtained in step (2) of example 1;

FIG. 3 is an EDS spectrum of the pDA/PN obtained in step (2);

FIG. 4 is a graph showing the hydrophilicity analysis of polycaprolactone, PN electrospun film obtained in step (1) and pDA/PN obtained in step (2) in example 1;

FIG. 5 is an SEM image of DFO@GMs in example 1;

FIG. 6 is an SEM image of a DFO@GMs-pDA/PN composite stent of example 1;

FIG. 7 is a graph showing degradation properties of the DFO@GMs-pDA/PN composite stent of example 1;

FIG. 8 is an SEM image of HUVECs after 48h of seeding on the surface of different scaffold materials;

FIG. 9 is a graph of the result of staining live/dead cells of the DFO@GMs-pDA/PN composite scaffold;

FIG. 10 is a graph showing the effect of DFO@GMs-pDA/PN composite scaffolds on cell proliferation activity;

FIG. 11 is a graph of the effect of DFO@GMs-pDA/PN composite scaffolds on HUVECs migration;

FIG. 12 is a graph showing the statistical analysis of the effect of the DFO@GMs-pDA/PN composite stent on HUVECs angiogenesis and the angiogenesis index, wherein graph A is a cell graph, and graphs B-D are the statistical analysis graphs of the total length, the number of connection points and the number of grids of the tube forming experiment;

FIG. 13 is a graph showing the effect of the DFO@GMs-pDA/PN composite scaffolds on the ability to promote osteogenic differentiation of BMSCs (blue alkaline phosphatase staining; red alizarin red staining);

FIG. 14 is a diagram showing the implantation of stents in different groupings during rat in vivo surgery;

FIG. 15 is a graph of the different stent materials implanted at 12 weeks of harvest;

FIG. 16 is a graph of the repair of a rat skull defect by Micro-CT scanning and 3D reconstruction;

FIG. 17 is a graph of the results of analysis of Bone Volume (BV), bone volume fraction (BV/TV), and trabecular bone separation (Tb.Sp) for a rat skull defect.

Detailed Description

In step (1) of the present invention, the electrospinning solution preferably comprises N, N-dimethylformamide and methylene chloride; the mass ratio of the N, N-dimethylformamide to the dichloromethane is preferably 3-4: 1, more preferably 3.2 to 3.5:1, a step of; the mass ratio of the nanoclay to the polycaprolactone to the electrospinning solvent is preferably 0.12-0.15: 1.5 to 2.5:8 to 10, more preferably 0.13 to 0.14:2 to 2.2:9 to 9.5.

In the step (1) of the present invention, the temperature of mixing is preferably 55 to 65 ℃, and more preferably 60 to 62 ℃; the stirring rate of the mixing is preferably 100 to 200r/min, more preferably 150 to 180r/min; the mixing time is preferably 50 to 60 minutes, more preferably 55 to 58 minutes.

In the step (1) of the present invention, parameters of the electrospinning are set as follows: the positive voltage is preferably 11.5 to 12.5kV, and more preferably 11.8 to 12.2kV; the negative voltage is preferably 2.3 to 2.6kV, and more preferably 2.4 to 2.5kV; the injection speed is preferably 0.14 to 0.16mm/min, and more preferably 0.15mm/min; the receiving distance is preferably 8 to 12cm, more preferably 9 to 10cm; the receiving speed is preferably 30 to 35r/min, and more preferably 32 to 34r/min; the receiving time is preferably 2.5 to 3 hours, more preferably 160 to 170 minutes.

In the step (1) of the invention, the specific steps of mixing the nanoclay, the polycaprolactone and the electrospinning solvent are as follows: mixing N, N-Dimethylformamide (DMF) and Dichloromethane (DCM), adding nanoclay (nanocrys NCs) thereto while stirring, magnetically stirring, and then placing in a water bath to perform ultrasonic dispersion to the solution to uniformly disperse the nanoclay; adding Polycaprolactone (PCL) thereto with stirring;

wherein the rotating speed of the magnetic stirring is preferably 100-200 r/min, and more preferably 150-180 r/min; the magnetic stirring time is preferably 5 to 8 minutes, more preferably 6 to 7 minutes; the ultrasonic frequency of the ultrasonic dispersion is preferably 80-120 kHz, more preferably 90-100 kHz; the ultrasonic dispersion time is preferably 10 to 15 minutes, more preferably 11 to 14 minutes; the temperature of the water bath is preferably 55 to 65℃and more preferably 60 to 62 ℃.

In the step (1), after the electrostatic spinning is finished, drying the obtained product in a fume hood, cutting the dried product into different sizes, and placing a bracket in a sample bag for normal temperature storage for later use; among them, the drying time is preferably 20 to 24 hours, more preferably 22 to 23 hours.

In the step (2) of the present invention, the mass ratio of the dopamine powder to the tris in the tris buffer is preferably 0.2:0.11 to 0.12, more preferably 0.2:0.115; the reaction time is preferably 10 to 14 hours, more preferably 12 to 13 hours; the stirring rate of the reaction is preferably 200 to 250r/min, more preferably 220 to 240r/min.

In the step (2) of the present invention, the preparation of the tris buffer solution comprises the steps of: mixing absolute ethanol and water, adding Tris (hydroxymethyl) -aminomethane (Tris), magnetically stirring to dissolve Tris particles, and adjusting the pH value of the solution by hydrochloric acid;

wherein, the mass volume ratio of the tris (hydroxymethyl) aminomethane, the absolute ethyl alcohol and the water is preferably 115-120 mg: 20-30 mL:80mL, more preferably 118 to 119mg: 25-28 mL:80mL; the rotation speed of the magnetic stirring is preferably 200-250 r/min, and more preferably 220-240 r/min; the magnetic stirring time is preferably 10 to 13 minutes, more preferably 11 to 12 minutes; the volume fraction of hydrochloric acid is preferably 10 to 15%, more preferably 12 to 14%; the amount of hydrochloric acid to be used is preferably such that the pH of the solution is 8.4 to 8.8, more preferably such that the pH of the solution is 8.5 to 8.6.

In the step (2), after the reaction is finished, filtering the obtained product, collecting the polydopamine modified PN stent main body by using a filter screen, flushing with water for 3 times to remove redundant polydopamine, and finally, naturally drying a sample overnight and collecting the sample in a sample bag.

In the step (3) of the invention, the mass-volume ratio of the gelatin, the deferoxamine and the water is preferably 2-3 g:0.01 to 0.02g:20 to 25mL, more preferably 2.5 to 2.8g:0.015g: 22-24 mL; the volume ratio of mineral oil to Span-80 is preferably 45-50: 1, more preferably 48 to 49:1, a step of; the volume ratio of Span-80 to glutaraldehyde is preferably 2:0.03 to 0.05, more preferably 2:0.04; the volume fraction of glutaraldehyde is preferably 45 to 50%, more preferably 46 to 48%.

In the step (3) of the present invention, the temperature of the crosslinking reaction is preferably 3 to 4 ℃, and more preferably 3.5 ℃; the time for the crosslinking reaction is preferably 20 to 30 minutes, more preferably 25 to 28 minutes.

In the step (3) of the present invention, the temperature of the mixing is independently preferably 60 to 65 ℃, and more preferably 62 to 64 ℃; the stirring rate of the mixture of gelatin, deferoxamine and water is preferably 30-40 r/min, more preferably 32-35 r/min; the stirring rate of mixing the mineral oil and Span-80 is preferably 1000 to 1500r/min, more preferably 1200 to 1300r/min.

In the step (3), after the crosslinking reaction is finished, washing the obtained product with acetone, centrifuging, and repeating the steps twice; freezing the obtained product in liquid nitrogen and then freeze-drying;

wherein the rotation speed of the centrifugation is preferably 5000-5500 r/min, and further preferably 5200-5300 r/min; the time of centrifugation is preferably 5 to 10 minutes, more preferably 6 to 8 minutes; the temperature of freeze drying is preferably-20 to-10 ℃, and more preferably-18 to-15 ℃; the time for freeze-drying is preferably 40 to 48 hours, more preferably 42 to 46 hours.

In the step (4) of the present invention, the mass-to-volume ratio of the DFO@GMs, ethanol and phosphate buffer salt solution is preferably 200mg: 47-50 mL:45 to 51mL, more preferably 200mg: 48-49 mL: 46-50 mL; the reaction time is preferably 10 to 14 hours, more preferably 12 to 13 hours; the stirring rate of the reaction is preferably 200 to 250r/min, more preferably 220 to 240r/min.

In the step (4), after the reaction is finished, centrifuging the obtained product, taking out a solid material, and air-drying to obtain the DFO@GMs-pDA/PN composite stent; wherein, the rotation speed of the centrifugation is preferably 5000-5500 r/min, and more preferably 5200-5300 r/min; the time for centrifugation is preferably 5 to 10 minutes, more preferably 6 to 8 minutes.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

(1) Preparation of PN (PCL/NCs) stent body:

8g of N, N-Dimethylformamide (DMF) and 2g of Dichloromethane (DCM) were weighed and mixed, and stirred magnetically to mix as an electrospinning solvent. 121mg of nanoclay (nanociys NCs) was weighed and added to the solvent with stirring, magnetically stirred for 5min, and then the solution was subjected to ultrasonic dispersion in a water bath for 10min to uniformly disperse the NCs in the solvent. Then 2g of Polycaprolactone (PCL) was weighed and added to the electrospinning solution with stirring, and the sample was rapidly transferred to a water bath at 60℃with magnetic stirring at 100r/min for about 60min until the PCL was completely dissolved, to prepare a sample of 1wt% NCs. And extracting the prepared electrospinning solution to prepare a PN electrospun film, wherein the electrospinning conditions are as follows: the positive voltage is 12kV, the negative voltage is 2.5kV, the injection speed is 0.15mm/min, the receiving distance is 10cm, the receiving speed is 30r/min, and the receiving time is 3h. And after the electrospinning is finished, marking information, drying for 24 hours in a fume hood, cutting into different sizes, and placing the bracket in a sample bag for normal temperature storage for standby.

(2) Polydopamine (pDA) surface modified scaffolds:

and the surface of the electrospun membrane is modified by utilizing the polymerization reaction of dopamine, so that the loading of the drug-loaded microspheres is facilitated. First, 20mL of absolute ethanol was measured and added to 80mL of deionized water for uniform mixing, 118mg of Tris (hydroxymethyl) -aminomethane (Tris) was weighed and added thereto, and Tris particles were dissolved by magnetic stirring for 10min. The solution was slowly dropped into Tris buffer using 10% dilute hydrochloric acid to adjust the pH to 8.6. Then 200mg of dopamine powder is weighed and added into the solution, and the solution is magnetically stirred at 200r/min, and simultaneously the cut electrospinning bracket is added for stirring to start the reaction. Stir overnight at room temperature, not completely close the beaker. After the reaction is completed, pDA/PN is collected by a filter screen, and the residual pDA is removed by washing 3 times with deionized water. Finally, the sample is collected in a sample bag after being naturally dried overnight.

(3) Preparation of dfo@gms:

the gelatin microsphere (Gelatin microspheres, GMs) coated with Deferoxamine (DFO) is prepared by adopting an emulsification-extraction method, namely DFO@GMs, and the preparation of the drug-loaded microsphere can be briefly described as the following process: and (3) solution A: 3g of gelatin is added into 20mL of deionized water, placed in a water bath at 60 ℃ and magnetically stirred for 10min at 30r/min until the gelatin is completely dissolved, then 20mgDFO is weighed and added into the gelatin solution, and stirring is continued for 10min. And (2) liquid B: 100ml of mineral oil was prepared and placed in a 60℃water bath with stirring at 1000r/min, 2ml span-80 was added dropwise thereto and stirred for 30min for sufficient emulsification. The solution A is added into the solution B dropwise at the speed of 1ml/min, the solution A is added while stirring, and stirring is continued for 20min after the completion until uniform emulsion is formed. The emulsion was then transferred into a 4℃ice bath and stirred at 1000r/min for 30min for cooling. Then 50. Mu.L of 50% glutaraldehyde was added to the emulsion and crosslinked by stirring at a constant speed in an ice bath for 20 min. 50mL ice-cold acetone was added thereto, followed by stirring at an original speed for 20min, washing the microspheres, followed by centrifugation at 5000r/min for 5min, and the supernatant was discarded. Then, 50mL of acetone was added, and after centrifugation at 5000r/min for 5min, the precipitate was collected, and the procedure was repeated 2 times. The microspheres were collected in a centrifuge tube, frozen in liquid nitrogen and lyophilized for 48h using a vacuum lyophilizer. And (5) filling the prepared microspheres into a centrifuge tube, and sealing and preserving at normal temperature.

(4) Assembling the DFO@GMs-pDA/PN composite stent:

50mL of absolute ethanol and 50mL of LPBS were weighed and mixed, 200mg of DFO@GMs was weighed and added to the solution, and pDA/PN was also added thereto and magnetically stirred at 200r/min overnight. And then collecting the solution and the bracket into a centrifuge tube, centrifuging for 5min at 5000r/min, taking out the bracket, and sealing for storage after water absorption and natural drying.

And (3) carrying out microscopic morphology analysis and EDS energy spectrum analysis on polycaprolactone, the PN electrospun film obtained in the step (1) and the pDA/PN obtained in the step (2) by using SEM, wherein the obtained results are shown in FIG. 2 and FIG. 3.

As can be seen from FIGS. 2 and 3, the invention successfully prepares the DFO@GMs-pDA/PN composite stent.

The hydrophilicities of polycaprolactone, PN electrospun film obtained in step (1) and pDA/PN obtained in step (2) were measured by static drop method, and the results are shown in FIG. 4.

As can be seen from FIG. 4, the higher hydrophilicity of pDA/PN is beneficial to enhance cell migration and enhance integration of the scaffold material with the host tissue.

The microscopic morphology analysis was performed on the DFO@GMs and the DFO@GMs-pDA/PN composite scaffolds by using SEM, and the obtained results are shown in FIG. 5 and FIG. 6.

As can be seen from FIGS. 5 and 6, the gelatin microsphere coated DFO on the DFO@GMs-pDA/PN composite stent is uniformly distributed on the matrix material of the stent.

The degradation performance of the dfo@gms-pDA/PN composite scaffold was tested by shaking incubation of the scaffold in an enzyme-containing neutral PBS solution using a formulation simulating the in vivo environment, and the results are shown in fig. 7.

Application example 1

The performance of the DFO@GMs-pDA/PN composite stent obtained in example 1 was examined, and the examination method and results were as follows.

(1) Biocompatibility of DFO@GMs-pDA/PN composite scaffold

The adhesion and morphology of HUVECs after 48h seeding on the surface of different scaffold materials were observed by SEM and the results are shown in fig. 8.

From fig. 8, it is clear that the cells on the surface of the dfo@gms-pDA/PN composite scaffold material are not only attached to the drug-loaded microsphere and electrospun fiber, but also more in number and more spread in morphology, thus forming a sheet-like fusion.

The cytotoxicity of the DFO@GMs-pDA/PN composite scaffold was evaluated by using live dead cell staining, and the results are shown in FIG. 9.

As can be seen from FIG. 9, the ratios of the live and dead cells in the different groups are not significantly different, and the characteristics of green fluorescence as the main component and red fluorescence as the least component are presented.

The effect of the DFO@GMs-pDA/PN composite scaffold on cell proliferation activity was examined by CCK-8 experiments, and the results are shown in FIG. 10.

As can be seen from fig. 10, all groups reached the highest proliferation activity on day 4, with no significant differences between groups at each time point.

The DFO@GMs-pDA/PN composite scaffold obtained by the invention has good biocompatibility, and can be used for normal adhesion, growth and proliferation of cells.

(2) Vasogenic Activity of DFO@GMs-pDA/PN composite stent

The effect of the composite scaffold on cell migration was examined using a scratch assay and the results obtained are shown in FIG. 11.

As can be seen from fig. 11, the composite scaffold can significantly promote the migration ability of HUVECs.

The effect and induction of in vitro composite scaffold materials on HUVECs angiogenesis was studied by an angiogenesis experiment, and the results are shown in fig. 12.

From fig. 12, it is clear that the addition of DFO has the ability to promote angiogenesis in HUVECs, and that the effect is more pronounced with increasing DFO dose (over a range), the DFO-20 group has the optimal effect of promoting angiogenesis. The release of DFO from the microsphere in DFO@GM endows the DFO@GMs-pDA/PN composite scaffold with the capability of promoting endothelial cell angiogenesis through the stimulation of HUVECs and the angiogenesis-related signal transmission.

(3) Osteogenic Activity of DFO@GMs-pDA/PN composite scaffold

The ability of the DFO@GMs-pDA/PN composite scaffold to promote osteogenic differentiation of BMSCs was examined by ALP and ARS staining, and the results are shown in FIG. 13.

From FIG. 13, it is clear that DFO release promotes early osteogenic differentiation (i.e., ALP expression) of rBMSCs, while NCs promote mineralization in the middle and late stages. The NCs loaded in the DFO@GMs-pDA/PN composite scaffold acts on rBMSCs in cooperation with the DFO, can effectively promote osteogenic differentiation of the rBMSCs, and has the potential of promoting bone tissue regeneration.

(4) In vivo osteogenic effect of DFO@GMs-pDA/PN composite scaffold

After a circular defect with the diameter of 5mm is constructed on the left side of a sagittal suture of a rat skull, a traditional BTE stent (Control), a PN stent main body obtained in the step (1) and a pDA/PN and DFO@GMs-pDA/PN composite stent obtained in the step (2) are implanted into a bone electrospinning defect. The results are shown in fig. 14 and 15.

As can be seen from fig. 14, the scaffold conforms to the size of the defect, and thus can be stably present at the defect to promote bone tissue regeneration. In the postoperative feeding process, all rats have good health condition, and wound healing is not obviously abnormal until the craniofacial skin is completely healed and hair grows normally when materials are obtained in 12 weeks. From fig. 15, it is known that the dfo@gms-pDA/PN composite scaffold (DFO-20) can exist stably in the defect area in vivo without displacement, and the scaffold is not disintegrated before complete bone healing, so that good support can be provided for tissue regeneration, and the basic requirements of tissue engineering technology on the regenerated scaffold are met.

The repair of the rat skull defect was analyzed using Micro-CT scan and 3D reconstruction to evaluate the repair effect of the DFO@GMs-pDA/PN composite scaffold on the skull defect, and the results are shown in FIG. 16.

As can be seen from FIG. 16, the DFO@GMs-pDA/PN composite scaffold can enable the defect area to be almost filled with new bone and has a certain thickness, and the rat cranium parietal bone is continuous.

The Bone Volume (BV), bone volume fraction (BV/TV), and bone trabecular separation (Tb.Sp) of the rat skull defect were analyzed, and the results are shown in FIG. 17.

As can be seen from FIG. 17, the DFO@GMs-pDA/PN composite scaffold had the largest average newly-formed bone volume, the largest bone mass change, i.e., the largest newly-formed bone and the reduced trabecular separation. The quantitative analysis result is consistent with the bone tissue regeneration degree shown by 3D reconstruction, which indicates that the loading of NCs and the loading of DFO@GMs lead the composite scaffold to have better effect of promoting bone healing in a rat body.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The preparation method of the DFO@GMs-pDA/PN composite stent for promoting vascularization and bone formation is characterized by comprising the following steps of:

2. The method for preparing a dfo@gms-pDA/PN composite scaffold for promoting vascularization and bone formation according to claim 1, wherein in the step (1), the electrospinning solution comprises N, N-dimethylformamide and dichloromethane; the mass ratio of the N, N-dimethylformamide to the dichloromethane is 3-4: 1, a step of; the mass ratio of the nanoclay to the polycaprolactone to the electrospinning solvent is 0.12-0.15: 1.5 to 2.5: 8-10.

3. The method for preparing a dfo@gms-pDA/PN composite scaffold for promoting vascularization and bone formation according to claim 2, wherein in the step (1), the mixing temperature is 55-65 ℃, the mixing stirring rate is 100-200 r/min, and the mixing time is 50-60 min.

4. A method for preparing a dfo@gms-pDA/PN composite stent for promoting vascularization and bone formation according to any one of claims 1 to 3, wherein in the step (1), parameters of electrospinning are set as follows: the positive voltage is 11.5-12.5 kV, the negative voltage is 2.3-2.6 kV, the injection speed is 0.14-0.16 mm/min, the receiving distance is 8-12 cm, the receiving speed is 30-35 r/min, and the receiving time is 2.5-3 h.

5. The method for preparing a dfo@gms-pDA/PN composite scaffold for promoting vascularization and bone formation according to claim 4, wherein in the step (2), the mass ratio of the dopamine powder to the tris buffer solution is 0.2:0.11 to 0.12; the reaction time is 10-14 h, and the stirring rate of the reaction is 200-250 r/min.

6. The method for preparing the DFO@GMs-pDA/PN composite scaffold for promoting vascularization and bone formation according to claim 5, wherein in the step (3), the mass-volume ratio of gelatin, deferoxamine and water is 2-3 g:0.01 to 0.02g: 20-25 mL; the volume ratio of the mineral oil to Span-80 is 45-50: 1, a step of; the volume ratio of Span-80 to glutaraldehyde is 2:0.03 to 0.05; the volume fraction of glutaraldehyde is 45-50%.

7. The method for preparing a dfo@gms-pDA/PN composite stent for promoting vascularization and bone formation according to claim 6, wherein in the step (3), the temperature of the crosslinking reaction is 3-4 ℃, and the time of the crosslinking reaction is 20-30 min.

8. The method for preparing a dfo@gms-pDA/PN composite stent for promoting vascularization and bone formation according to claim 1, 5, 6 or 7, wherein in the step (4), the mass-to-volume ratio of dfo@gms, ethanol and phosphate buffer salt solution is 200mg: 47-50 mL: 45-51 mL; the reaction time is 10-14 h, and the stirring rate of the reaction is 200-250 r/min.

9. The dfo@gms-pDA/PN composite stent prepared by the method for preparing the dfo@gms-pDA/PN composite stent for promoting vascularization and bone formation according to any one of claims 1 to 8.

10. Use of the dfo@gms-pDA/PN composite scaffold of claim 9 in scaffolds for bone tissue engineering.