DFO@GMs-pDA / PN composite scaffold for promoting vascularization and osteogenesis, and preparation method and application thereof

Abstract

The application belongs to the field of biomedical materials and biomedical engineering technology, and discloses a DFO@GMs-pDA / PN composite scaffold for promoting vascularization and osteogenesis, and a preparation method and application thereof. The DFO@GMs-pDA / PN composite scaffold for promoting vascularization and osteogenesis is constructed by using electrospinning, pDA modification and emulsification-extraction technology, and by wrapping DFO into GMs and loading the DFO into a pDA surface modified PN electrospinning film. The most superficial DFO@GMs begin to disintegrate and release DFO after implantation, and the maximum drug release rate is reached after 48 hours, so as to promote the ingrowth 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 to provide an important blood supply basis for bone regeneration.

Description

Technical Field

[0001] This invention relates to the fields of biomedical materials and biomedical engineering technology, and in particular to a DFO@GMs-pDA / PN composite scaffold that promotes vascularization and osteogenic formation, its preparation method, and its application. Background Technology

[0002] Bone tissue is a complex matrix composed of a mineral phase that provides mechanical strength and osteoconductive matrix, and a collagen phase that plays a central role in progenitor cell differentiation and mineralization maturation. Simultaneously, bone is a highly vascularized tissue, relying on the connections between blood vessels and osteocytes to maintain its integrity. Bone regeneration is a complex, diverse, and coordinated process, with the coupling of angiogenesis and osteoblasts being crucial. The vascular network not only serves as a pathway for recruiting osteoprogenitors and endothelial progenitors, but also ensures sufficient oxygen and nutrients in bone tissue and facilitates the timely removal of metabolic waste. Furthermore, it participates in the regulation of various cells and signaling molecules during bone regeneration, maintaining the internal homeostasis of bone tissue. Vascularized bone regeneration is key to ensuring complete bone healing, accelerating the bone repair process, and improving the quality of bone remodeling. Achieving osteoblast-angiogenesis coupling during bone healing is a core challenge currently addressed in bone tissue engineering (BTE) technology.

[0003] In recent years, the scaffolds designed in bone tissue regeneration research have mainly adopted technologies such as electrospinning, hydrogels, freeze-drying, and 3D printing. Among them, electrospinning is a technology that uses electrostatic force to prepare fine fibers from polymer solutions. It has advantages such as extremely high surface area-to-volume ratio, adjustable porosity, extensibility to adapt to various sizes and shapes, and controllability of nanofiber composition. When used in BTE, electrospinning technology has the following advantages: (1) its structure can simulate the extracellular matrix (ECM) of bone tissue; (2) its high porosity and permeability facilitate the diffusion of nutrients, which is beneficial to cell growth, proliferation, and migration; (3) it has a high loading efficiency for bioactive factors, peptides, or drugs. PCL has been approved by the FDA for use in biomedicine. It has suitable tensile properties, biocompatibility, osseointegration, and biodegradability. Moreover, its degradation rate is appropriate. It will not degrade too quickly to support tissue regeneration, nor will it degrade too slowly to affect tissue regeneration. Therefore, it is a suitable scaffold material for BTE. However, PCL itself is highly hydrophobic, which tends to inhibit cell migration and delay the integration of scaffold materials with host tissues. Therefore, pure PCL scaffolds exhibit poor cell adhesion and lack bioactivity, resulting in a lack of biological function at the scaffold-cell interface. Nanomaterials (NCs) are two-dimensional nanomaterials for bone tissue engineering with multiple functions, such as intrinsic osteoinduction, improved mechanical properties, and enhanced drug release capabilities. 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, promoting water adsorption and hydrolytic degradation of PCL chains, thus facilitating cell adhesion, proliferation, and migration. Simultaneously, NCs can increase the roughness of electrospun fibers and have been shown to possess potential osteoinductive properties and promote biomineralization. Furthermore, NCs also possess high specific surface area and charge anisotropy, exhibiting strong drug binding and sustained-release capabilities for various molecules.

[0004] Microspheres are spherical particles that allow drug molecules to disperse within them, encapsulating and carrying the drug with their extremely small backbone. Gelatin is a product of collagen degradation in animal connective tissue or epidermal tissue. It is a linear polymer formed by cross-linking 18 amino acids with polypeptides, exhibiting low antigenicity, good degradability, and biocompatibility. Its molecular structure is also rich in arginine-glycine-aspartic acid sequences that promote cell adhesion and migration.

[0005] DFO promotes angiogenesis-osteogenic coupling mainly through the following pathways: (1) In the early stages of bone regeneration, DFO first activates the HIF-1α signaling pathway to promote the expression of a series of angiogenesis signals and active factors, thereby promoting the formation of new blood vessels in the target tissue. These new blood vessels serve as necessary channels for establishing connections between the defect area and adjacent tissues. While recruiting osteoprogenitor cells or osteoblast precursor cells for tissue regeneration, they also provide nutrients and oxygen to seed cells and transport waste products to support the growth, migration, and differentiation of stem cells. (2) DFO has been shown to promote the secretion of BMP-2 by endothelial cells to promote osteogenic differentiation of mesenchymal stem cells; and under this stimulation, mesenchymal stem cells can secrete VEGF to promote angiogenesis, thus achieving angiogenesis-osteogenic coupling through the interaction of the two types of cells. (3) DFO can inhibit the differentiation of osteoclast precursor cells in remodeled / reconstructed bone tissue, indirectly promoting osteogenic formation by preventing bone loss. Past studies have shown that the release time of DFO after physical adsorption onto the surface of scaffold materials is about 8 hours, which cannot achieve the effect of targeted application and controlled sustained release.

[0006] Traditional BTE stents have limitations due to the difficulty in preparation, easy inactivation, inability to be targeted, and side effects of the angiogenic / osteogenic active factors they carry or add. Summary of the Invention

[0007] The purpose of this invention is to provide a DFO@GMs-pDA / PN composite scaffold that promotes angiogenesis and osteogenic formation, as well as its preparation method and application, to solve the limitations of traditional BTE scaffolds in carrying or adding angiogenic / osteogenic active factors, such as difficulty in preparation, easy inactivation, inability to target application, and side effects.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] This invention provides a method for preparing a DFO@GMs-pDA / PN composite scaffold that promotes vascularization and osteogenic formation, comprising the following steps:

[0010] (1) Preparation of PN scaffold body: Nano clay, polycaprolactone and electrospinning solvent are mixed to obtain a mixture; the mixture is electrospinned to obtain PN scaffold body;

[0011] (2) Preparation of polydopamine-modified PN scaffold body: Dopamine powder and tris(hydroxymethyl)aminomethane buffer were mixed to obtain a mixture; the PN scaffold body was immersed in the mixture to react and obtain polydopamine-modified PN scaffold body;

[0012] (3) Preparation of DFO@GMs: Gelatin, deferoxamine and water were mixed to obtain solution A; mineral oil and Span-80 were mixed to obtain solution B; solution A, solution B and glutaraldehyde were cross-linked to obtain DFO@GMs;

[0013] (4) Preparation of DFO@GMs-pDA / PN composite scaffold: DFO@GMs, ethanol and phosphate buffer solution were mixed to obtain a mixture; the polydopamine modified PN scaffold body was immersed in the mixture to react and obtain DFO@GMs-pDA / PN composite scaffold.

[0014] Preferably, in step (1), the electrospinning solution includes N,N-dimethylformamide and dichloromethane; the mass ratio of N,N-dimethylformamide and dichloromethane is 3-4:1; and the mass ratio of nano-clay, polycaprolactone and electrospinning solvent is 0.12-0.15:1.5-2.5:8-10.

[0015] Preferably, in step (1), the mixing temperature is 55-65°C, the mixing speed is 100-200 r / min, and the mixing time is 50-60 min.

[0016] Preferably, in step (1), the electrospinning parameters are set as follows: positive voltage is 11.5-12.5kV, negative voltage is 2.3-2.6kV, injection speed is 0.14-0.16mm / min, receiving distance is 8-12cm, receiving speed is 30-35r / min, and receiving time is 2.5-3h.

[0017] Preferably, in step (2), the mass ratio of dopamine powder to tris(hydroxymethyl)aminomethane in the tris(hydroxymethyl)aminomethane buffer is 0.2:0.11-0.12; the reaction time is 10-14 h; and the stirring rate is 200-250 r / min.

[0018] Preferably, in step (3), the mass-to-volume ratio of gelatin, deferoxamine, and water is 2-3 g: 0.01-0.02 g: 20-25 mL; the volume ratio of mineral oil and Span-80 is 45-50:1; the volume ratio of Span-80 and glutaraldehyde is 2:0.03-0.05; and the volume fraction of glutaraldehyde is 45-50%.

[0019] Preferably, in step (3), the crosslinking reaction temperature is 3-4°C and the crosslinking reaction time is 20-30 min.

[0020] Preferably, in step (4), the mass-to-volume ratio of DFO@GMs, ethanol and phosphate buffer solution is 200 mg: 47-50 mL: 45-51 mL; the reaction time is 10-14 h; and the stirring rate is 200-250 r / min.

[0021] The present invention also provides a method for preparing the DFO@GMs-pDA / PN composite scaffold that promotes vascularization and osteogenic formation, resulting in the DFO@GMs-pDA / PN composite scaffold.

[0022] The present invention also provides the application of the DFO@GMs-pDA / PN composite scaffold in scaffolds used in bone tissue engineering.

[0023] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] (1) The DFO used in this invention, as a small molecule drug, can promote a complex angiogenesis process involving multiple factors, multiple pathways, and multiple active factors by inhibiting prolyl hydroxylase-domain proteins (PHDs) and activating the hypoxia-inducible factor-1α (HIF-1α) signaling pathway. Compared with the limitations of previous studies that only delivered 1 to 2 growth factors, DFO can also couple with osteogenic formation after promoting angiogenesis, thus maximizing bone healing during tissue regeneration.

[0025] (2) This invention utilizes electrospinning and emulsification-extraction technologies, using a PN electrospun membrane as the main body of a composite scaffold, and combining drug-loaded microspheres DFO@GMs with the PN electrospun scaffold through pDA surface modification. This produces a composite scaffold, DFO@GMs-pDA / PN, capable of locally releasing angiogenic drugs and osteogenic factors, with the two working synergistically to couple angiogenesis and osteogenicity, thereby promoting vascularized bone regeneration. The outermost DFO@GMs begins to disintegrate and release DFO after implantation, reaching its maximum release rate after 48 hours. In the early stages of bone regeneration, it promotes the ingrowth of new blood vessels, the recruitment of stem cells, and the secretion and presentation of related factors, providing an important blood supply basis for bone regeneration. Meanwhile, the PN electrospun scaffold, whose performance matches the bone structure of the defect model, can stably exist in the defect area during bone regeneration, mimicking the extracellular matrix of bone tissue, providing spatial support for stem cell proliferation and differentiation and new tissue formation, promoting osteogenic differentiation and subsequent mineralization, and effectively promoting bone regeneration.

[0026] (3) The composite scaffold modified with pDA and loaded with DFO@GMs exhibits low cytotoxicity and can, to a certain extent, support normal cell adhesion, growth, and proliferation. Its cell compatibility suggests that the scaffold material possesses the basic characteristics of a tissue engineering scaffold. In addition, the release of DFO from the microspheres in DFO@GMs, through stimulation of human umbilical vein endothelial cells (HUVECs) and angiogenesis-related signal transduction, endows the DFO@GMs-pDA / PN composite scaffold with the ability to promote endothelial cell angiogenesis. At the same time, the NCs loaded in the DFO@GMs-pDA / PN composite scaffold, in synergy with DFO, act on rat bone marrow mesenchymal stem cells (rBMSCs), effectively promoting osteogenic differentiation of rBMSCs and demonstrating the potential to promote bone tissue regeneration.

[0027] (4) An in vivo study was conducted by constructing a rat skull defect repair model and implanting a DFO@GMs-pDA / PN composite scaffold. The results showed that the implanted DFO@GMs-pDA / PN composite scaffold could stably exist in the bone defect area, exhibiting good in vivo biocompatibility. It also facilitated the healing and restoration of the continuity of the damaged periosteum, maintained the osteogenic space, and supported the regeneration of bone tissue in the defect area, significantly promoting the repair of rat skull defects. The newly formed bone tissue had the same structure as the original bone tissue and possessed mature lamellar bone, maintaining brain space and providing protection for soft tissue and supporting the skull morphology. This indicates that the DFO@GMs-pDA / PN composite scaffold obtained in this invention can effectively promote vascularized bone regeneration of the skull and accelerate the repair of bone defects in rats through angiogenesis-coupled osteogenic processes. It is a promising BTE scaffold, providing a new direction for bone regeneration research and clinical treatment of bone defects. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0029] Figure 1 A schematic diagram of the fabrication process of the DFO@GMs-pDA / PN composite scaffold;

[0030] Figure 2 SEM images of polycaprolactone, the PN electrospun film obtained in step (1), and the pDA / PN obtained in step (2) in Example 1;

[0031] Figure 3 The EDS energy spectrum of pDA / PN obtained in step (2);

[0032] Figure 4 The hydrophilicity analysis diagrams are of polycaprolactone, the PN electrospun film obtained in step (1), and the pDA / PN obtained in step (2) in Example 1.

[0033] Figure 5 This is a SEM image of DFO@GMs in Example 1;

[0034] Figure 6 The image shows the SEM image of the DFO@GMs-pDA / PN composite stent in Example 1.

[0035] Figure 7 The graph shows the degradation performance of the DFO@GMs-pDA / PN composite scaffold in Example 1.

[0036] Figure 8 SEM images of HUVECs after 48 hours of inoculation onto different scaffold materials;

[0037] Figure 9 Image showing the results of live / dead cell staining on the DFO@GMs-pDA / PN composite scaffold;

[0038] Figure 10 Figure showing the effect of DFO@GMs-pDA / PN composite scaffold on cell proliferation activity;

[0039] Figure 11 Figure showing the effect of DFO@GMs-pDA / PN composite scaffold on HUVEC migration;

[0040] Figure 12 Figure A shows the effect of DFO@GMs-pDA / PN composite scaffold on angiogenesis in HUVECs and the statistical analysis of angiogenesis indicators. Figure B to D are statistical analysis of the total length of the tube formation experiment, the number of connection points, and the number of grids.

[0041] Figure 13 The effect of the DFO@GMs-pDA / PN composite scaffold on the ability to promote osteogenic differentiation of BMSCs is shown in the figure (blue is the alkaline phosphatase staining figure; red is the alizarin red staining figure).

[0042] Figure 14 Figures showing the implantation of scaffolds in different groups of rats during surgery;

[0043] Figure 15 Images of different stent materials implanted at 12 weeks of gestation;

[0044] Figure 16This image shows the repair of skull defects in rats using Micro-CT scanning and 3D reconstruction.

[0045] Figure 17 The figure shows the analysis results of bone volume (BV), bone volume fraction (BV / TV), and trabecular separation (Tb.Sp) in rat skull defects. Detailed Implementation

[0046] This invention provides a method for preparing a DFO@GMs-pDA / PN composite scaffold that promotes vascularization and osteogenic formation, comprising the following steps:

[0047] (1) Preparation of PN scaffold body: Nano clay, polycaprolactone and electrospinning solvent are mixed to obtain a mixture; the mixture is electrospinned to obtain PN scaffold body;

[0048] (2) Preparation of polydopamine-modified PN scaffold body: Dopamine powder and tris(hydroxymethyl)aminomethane buffer were mixed to obtain a mixture; the PN scaffold body was immersed in the mixture to react and obtain polydopamine-modified PN scaffold body;

[0049] (3) Preparation of DFO@GMs: Gelatin, deferoxamine and water were mixed to obtain solution A; mineral oil and Span-80 were mixed to obtain solution B; solution A, solution B and glutaraldehyde were cross-linked to obtain DFO@GMs;

[0050] (4) Preparation of DFO@GMs-pDA / PN composite scaffold: DFO@GMs, ethanol and phosphate buffer solution were mixed to obtain a mixture; the polydopamine modified PN scaffold body was immersed in the mixture to react and obtain DFO@GMs-pDA / PN composite scaffold.

[0051] In step (1) of the present invention, the electrospinning solution preferably includes N,N-dimethylformamide and dichloromethane; the mass ratio of N,N-dimethylformamide and dichloromethane is preferably 3-4:1, more preferably 3.2-3.5:1; the mass ratio of nano-clay, polycaprolactone and electrospinning solvent is preferably 0.12-0.15:1.5-2.5:8-10, more preferably 0.13-0.14:2-2.2:9-9.5.

[0052] In step (1) of the present invention, the mixing temperature is preferably 55-65°C, more preferably 60-62°C; the mixing stirring rate is preferably 100-200 r / min, more preferably 150-180 r / min; and the mixing time is preferably 50-60 min, more preferably 55-58 min.

[0053] In step (1) of this invention, the parameters for electrospinning are set as follows: the positive voltage is preferably 11.5-12.5 kV, more preferably 11.8-12.2 kV; the negative voltage is preferably 2.3-2.6 kV, more preferably 2.4-2.5 kV; the injection speed is preferably 0.14-0.16 mm / min, more preferably 0.15 mm / min; the receiving distance is preferably 8-12 cm, more preferably 9-10 cm; the receiving speed is preferably 30-35 r / min, more preferably 32-34 r / min; and the receiving time is preferably 2.5-3 h, more preferably 160-170 min.

[0054] In step (1) of this invention, the specific steps for mixing nanoclay, polycaprolactone, and electrospinning solvent are as follows: N,N-dimethylformamide (DMF) and dichloromethane (DCM) are mixed, and nanoclay (Nanoclay NCs) are added to it while stirring. After magnetic stirring, the solution is placed in a water bath for ultrasonic dispersion to ensure uniform dispersion of the nanoclay; then polycaprolactone (PCL) is added to it while stirring.

[0055] The preferred speed of the magnetic stirring is 100-200 r / min, more preferably 150-180 r / min; the preferred stirring time is 5-8 min, more preferably 6-7 min; the preferred ultrasonic frequency for ultrasonic dispersion is 80-120 kHz, more preferably 90-100 kHz; the preferred ultrasonic dispersion time is 10-15 min, more preferably 11-14 min; and the preferred water bath temperature is 55-65℃, more preferably 60-62℃.

[0056] In step (1) of the present invention, after electrospinning, the obtained product is dried in a fume hood, and after drying, it is cut into different sizes and the support is placed in a sample bag and stored at room temperature for later use; wherein, the drying time is preferably 20-24h, and more preferably 22-23h.

[0057] In step (2) of the present invention, the mass ratio of dopamine powder to tris(hydroxymethyl)aminomethane in the buffer solution is preferably 0.2:0.11-0.12, more preferably 0.2:0.115; the reaction time is preferably 10-14 h, more preferably 12-13 h; the stirring rate of the reaction is preferably 200-250 r / min, more preferably 220-240 r / min.

[0058] In step (2) of the present invention, the preparation of the tris(hydroxymethyl)aminomethane buffer solution includes the following steps: mixing anhydrous ethanol and water, adding tris(hydroxymethyl)aminomethane (Tris), dissolving the Tris particles by magnetic stirring, and then adjusting the pH of the solution using hydrochloric acid.

[0059] The preferred mass-to-volume ratio of tris(hydroxymethyl)aminomethane, anhydrous ethanol, and water is 115–120 mg: 20–30 mL: 80 mL, more preferably 118–119 mg: 25–28 mL: 80 mL; the preferred magnetic stirring speed is 200–250 r / min, more preferably 220–240 r / min; the preferred magnetic stirring time is 10–13 min, more preferably 11–12 min; the preferred volume fraction of hydrochloric acid is 10–15%, more preferably 12–14%; and the preferred amount of hydrochloric acid is to make the pH of the above solution 8.4–8.8, more preferably 8.5–8.6.

[0060] In step (2) of the present invention, after the reaction is completed, the obtained product is filtered, the polydopamine-modified PN scaffold body is collected with a filter screen, and then rinsed with water three times to remove excess polydopamine. Finally, the sample is dried naturally overnight and collected in a sample bag.

[0061] In step (3) of the present invention, the preferred mass-to-volume ratio of gelatin, deferoxamine, and water is 2-3 g: 0.01-0.02 g: 20-25 mL, more preferably 2.5-2.8 g: 0.015 g: 22-24 mL; the preferred volume ratio of mineral oil and Span-80 is 45-50:1, more preferably 48-49:1; the preferred volume ratio of Span-80 and glutaraldehyde is 2:0.03-0.05, more preferably 2:0.04; and the preferred volume fraction of glutaraldehyde is 45-50%, more preferably 46-48%.

[0062] In step (3) of the present invention, the temperature of the crosslinking reaction is preferably 3-4°C, more preferably 3.5°C; the time of the crosslinking reaction is preferably 20-30 min, more preferably 25-28 min.

[0063] In step (3) of the present invention, the mixing temperature is preferably 60-65°C, more preferably 62-64°C; the stirring rate for mixing gelatin, deferoxamine and water is preferably 30-40 r / min, more preferably 32-35 r / min; the stirring rate for mixing mineral oil and Span-80 is preferably 1000-1500 r / min, more preferably 1200-1300 r / min.

[0064] In step (3) of the present invention, after the crosslinking reaction is completed, the obtained product is washed with acetone and centrifuged, and the above steps are repeated twice; the obtained product is frozen in liquid nitrogen and then freeze-dried.

[0065] The centrifugation speed is preferably 5000-5500 r / min, more preferably 5200-5300 r / min; the centrifugation time is preferably 5-10 min, more preferably 6-8 min; the freeze-drying temperature is preferably -20--10℃, more preferably -18--15℃; and the freeze-drying time is preferably 40-48 h, more preferably 42-46 h.

[0066] In step (4) of the present invention, the preferred mass-to-volume ratio of DFO@GMs, ethanol and phosphate buffer solution is 200 mg: 47-50 mL: 45-51 mL, more preferably 200 mg: 48-49 mL: 46-50 mL; the preferred reaction time is 10-14 h, more preferably 12-13 h; the preferred stirring rate is 200-250 r / min, more preferably 220-240 r / min.

[0067] In step (4) of the present invention, after the reaction is completed, the obtained product is centrifuged, and then the solid material is taken out and air-dried to obtain DFO@GMs-pDA / PN composite scaffold; wherein, the centrifugation speed is preferably 5000-5500 r / min, more preferably 5200-5300 r / min; the centrifugation time is preferably 5-10 min, more preferably 6-8 min.

[0068] The present invention also provides a method for preparing the DFO@GMs-pDA / PN composite scaffold that promotes vascularization and osteogenic formation, resulting in the DFO@GMs-pDA / PN composite scaffold.

[0069] The present invention also provides the application of the DFO@GMs-pDA / PN composite scaffold in scaffolds used in bone tissue engineering.

[0070] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0071] Example 1

[0072] (1) Fabrication of the PN(PCL / NCs) scaffold body:

[0073] Weigh 8g of N,N-dimethylformamide (DMF) and 2g of dichloromethane (DCM) and mix them, stirring magnetically to obtain the electrospinning solvent. Weigh 121mg of nanoclay (NCs) and add it to the solvent while stirring. After stirring magnetically for 5 minutes, place the solution in a water bath and sonicate for 10 minutes to ensure uniform dispersion of NCs in the solvent. Then weigh 2g of polycaprolactone (PCL) and add it to the electrospinning solution while stirring. Quickly transfer the sample to a 60℃ water bath and stir magnetically at 100r / min for about 60 minutes until the PCL is completely dissolved, preparing a 1wt% NCs sample. The obtained electrospinning solution was extracted to prepare PN electrospinned membranes. The electrospinning conditions were as follows: positive voltage 12kV, negative voltage 2.5kV, injection speed 0.15mm / min, receiving distance 10cm, receiving speed 30r / min, and receiving time 3h. After electrospinning, the membranes were labeled, dried in a fume hood for 24h, cut into different sizes, and placed in sample bags for storage at room temperature for later use.

[0074] (2) Polydopamine (pDA) surface-modified scaffold:

[0075] The surface of the electrospun membrane was modified using the polymerization of dopamine to facilitate the loading of drug-loaded microspheres. First, 20 mL of anhydrous ethanol was added to 80 mL of deionized water and mixed thoroughly. Then, 118 mg of tris-(hydroxymethyl)-aminomethane (Tris) was added and the mixture was magnetically stirred for 10 min to dissolve the Tris particles. The pH of the solution was adjusted to 8.6 by slowly adding 10% dilute hydrochloric acid dropwise to the Tris buffer. Next, 200 mg of dopamine powder was added to the solution, and the mixture was magnetically stirred at 200 rpm while simultaneously adding a pre-cut electrospun scaffold to initiate the reaction. The mixture was stirred overnight at room temperature, with the beaker not completely sealed. After the reaction was complete, pDA / PN was collected using a filter and rinsed three times with deionized water to remove excess pDA. Finally, the sample was allowed to air dry overnight and collected in a sample bag.

[0076] (3) Preparation of DFO@GMs:

[0077] Gelatin microspheres (GMs) encapsulating deferoxamine (DFO) were prepared using an emulsification-extraction method. The preparation process can be summarized as follows: Solution A: 3g of gelatin was added to 20mL of deionized water and placed in a 60℃ water bath with magnetic stirring at 30r / min for 10min until the gelatin was completely dissolved. Then, 20mg of DFO was weighed and added to the gelatin solution, and stirring was continued for 10min. Solution B: 100ml of mineral oil was prepared and placed in a 60℃ water bath with stirring at 1000r / min. 2ml of Span-80 was added dropwise and stirred for 30min to ensure complete emulsification. Solution A was added dropwise to Solution B at a rate of 1ml / min while stirring. After completion, stirring was continued for 20min until a homogeneous emulsion was formed. The emulsion was then transferred to a 4℃ ice bath and stirred at 1000r / min for 30min to cool. Then, 50 μL of 50% glutaraldehyde was added to the emulsion, and crosslinking was carried out by stirring at the same speed for 20 min in an ice bath. 50 mL of ice-cold acetone was added, and the mixture was stirred at the original speed for 20 min to wash the microspheres. The microspheres were then centrifuged at 5000 rpm for 5 min, and the supernatant was discarded. Another 50 mL of acetone was added, and the mixture was centrifuged at 5000 rpm for 5 min, and the precipitate was collected. This operation was repeated twice. The microspheres were collected in centrifuge tubes, frozen in liquid nitrogen, and then freeze-dried using a vacuum freeze dryer for 48 h. The prepared microspheres were then stored in sealed centrifuge tubes at room temperature.

[0078] (4) Assembly of DFO@GMs-pDA / PN composite stent:

[0079] Measure and mix 50 mL of anhydrous ethanol and 50 mL of PBS. Weigh 200 mg of DFO@GMs and add it to the solution, along with pDA / PN. Stir magnetically at 200 rpm overnight. Then, collect the solution and the support in a centrifuge tube, centrifuge at 5000 rpm for 5 min, remove the support, absorb water, allow it to air dry, and then seal and store it for later use.

[0080] Microstructure analysis of polycaprolactone, the PN electrospun film obtained in step (1), and the pDA / PN obtained in step (2) were performed using SEM and EDS. The results are as follows. Figure 2 and Figure 3 As shown.

[0081] Depend on Figure 2 and Figure 3 It can be seen that the present invention has successfully prepared a DFO@GMs-pDA / PN composite scaffold.

[0082] The hydrophilicity of polycaprolactone, the PN electrospun film obtained in step (1), and the pDA / PN obtained in step (2) was determined using the static droplet method. The results are as follows: Figure 4 As shown.

[0083] Depend on Figure 4 It is known that pDA / PN has high hydrophilicity, which is beneficial for improving cell migration and enhancing the integration of scaffold materials with host tissues.

[0084] Microstructure analysis of DFO@GMs and DFO@GMs-pDA / PN composite scaffolds was performed using SEM, and the results are as follows: Figure 5 and Figure 6 As shown.

[0085] Depend on Figure 5 and Figure 6 It can be seen that the gelatin microspheres encapsulating DFO are uniformly distributed on the matrix material of the DFO@GMs-pDA / PN composite scaffold.

[0086] The DFO@GMs-pDA / PN composite scaffold was immersed in an enzyme-containing neutral PBS solution and incubated with shaking using a formulation that simulates the in vivo environment. Its degradation performance was then assessed, and the results are as follows: Figure 7 As shown.

[0087] Application Example 1

[0088] The performance of the DFO@GMs-pDA / PN composite stent obtained in Example 1 was tested, and the test methods and results are as follows.

[0089] (1) Biocompatibility of DFO@GMs-pDA / PN composite scaffold

[0090] The adhesion and morphology of HUVECs seeded on different scaffold materials for 48 hours were observed using SEM. The results are as follows: Figure 8 As shown.

[0091] Depend on Figure 8 It can be seen that the cells on the surface of the DFO@GMs-pDA / PN composite scaffold material not only adhere to the drug-loaded microspheres and electrospun fibers, but also have a greater number and more spread-out morphology, forming a sheet-like fusion.

[0092] The cytotoxicity of the DFO@GMs-pDA / PN composite scaffold was evaluated using live-dead cell staining, and the results are as follows: Figure 9 As shown.

[0093] Depend on Figure 9 It can be seen that there is no significant difference in the ratio of live to dead cells among different groups, and all groups show the characteristics of green fluorescence as the main component and red fluorescence as a very small component.

[0094] The effect of the DFO@GMs-pDA / PN composite scaffold on cell proliferation was detected by CCK-8 assay, and the results are as follows: Figure 10 As shown.

[0095] Depend on Figure 10 It can be seen that all groups reached their highest proliferation activity on day 4, and there was no significant difference between the groups at each time point.

[0096] The DFO@GMs-pDA / PN composite scaffold obtained in this invention has good biocompatibility and can facilitate normal cell adhesion, growth and proliferation.

[0097] (2) Angiogenic activity of DFO@GMs-pDA / PN composite stent

[0098] The effect of the composite scaffold on cell migration was detected using a scratch assay, and the results are as follows: Figure 11 As shown.

[0099] Depend on Figure 11 It can be seen that composite scaffolds can significantly promote the migration ability of HUVECs.

[0100] Angiogenesis experiments were conducted to investigate the effects and induction of angiogenesis in HUVECs by in vitro composite scaffold materials. The results are as follows: Figure 12 As shown.

[0101] Depend on Figure 12 It is evident that the addition of DFO promotes angiogenesis in HUVECs, and the effect becomes more pronounced with increasing DFO dosage (within a certain range), with the DFO-20 group exhibiting the best angiogenesis-promoting effect. The release of DFO from the microspheres in DFO@GM, through stimulation of HUVECs and angiogenesis-related signal transduction, endows the DFO@GMs-pDA / PN composite scaffold with the ability to promote endothelial cell angiogenesis.

[0102] (3) Osteogenic activity of DFO@GMs-pDA / PN composite scaffold

[0103] The ability of the DFO@GMs-pDA / PN composite scaffold to promote osteogenic differentiation of BMSCs was assessed using ALP and ARS staining. The results are as follows: Figure 13 As shown.

[0104] Depend on Figure 13 It is known that DFO release can promote early osteogenic differentiation of rBMSCs (i.e., ALP expression), while NCs promote mid-to-late-stage mineralization. NCs loaded in the DFO@GMs-pDA / PN composite scaffold synergistically act on rBMSCs with DFO, effectively promoting osteogenic differentiation of rBMSCs and demonstrating the potential to promote bone tissue regeneration.

[0105] (4) In vivo osteogenic effect of DFO@GMs-pDA / PN composite scaffold

[0106] After constructing a 5mm diameter circular defect on the left side of the sagittal suture in the rat skull, a traditional BTE scaffold (Control), the PN scaffold body obtained in step (1), the pDA / PN obtained in step (2), and the DFO@GMs-pDA / PN composite scaffold were implanted into the bone electrospinning defect. The results are as follows. Figure 14 and Figure 15 As shown.

[0107] Depend on Figure 14 It can be seen that the scaffold size matches the defect, thus it can stably exist at the defect site and promote bone tissue regeneration. During postoperative care, all rats were in good health, and wound healing showed no obvious abnormalities until 12 weeks when the skull skin was completely healed and hair grew normally. Figure 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 does not disintegrate before complete bone healing, thus providing good support for tissue regeneration and meeting the basic requirements of tissue engineering technology for regenerative scaffolds.

[0108] Micro-CT scanning and 3D reconstruction were used to analyze the repair of skull defects in rats to evaluate the repair effect of the DFO@GMs-pDA / PN composite scaffold on skull defects. The results are as follows: Figure 16 As shown.

[0109] Depend on Figure 16 It can be seen that the DFO@GMs-pDA / PN composite scaffold can almost fill the defect area with new bone and have a certain thickness, and the skull bone of rats is continuous.

[0110] Bone volume (BV), bone volume fraction (BV / TV), and trabecular bone separation (Tb.Sp) of rat skull defects were analyzed, and the results are as follows: Figure 17 As shown.

[0111] Depend on Figure 17 The results showed that the DFO@GMs-pDA / PN composite scaffold exhibited the highest average volume of new bone formation, the greatest change in bone mass (i.e., the most new bone formation), and reduced trabecular separation in the bone defect area. Quantitative analysis results were consistent with the degree of bone regeneration shown in the 3D reconstruction, suggesting that the loading of NCs and DFO@GMs enabled the composite scaffold to effectively promote bone healing in rats.

[0112] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a DFO@GMs-pDA / PN composite scaffold that promotes vascularization and osteogenic formation, characterized in that, Includes the following steps: (1) Preparation of PN scaffold body: Nano clay, polycaprolactone and electrospinning solvent are mixed to obtain a mixture; the mixture is electrospinned to obtain PN scaffold body; (2) Preparation of polydopamine-modified PN scaffold body: Dopamine powder and tris(hydroxymethyl)aminomethane buffer were mixed to obtain a mixture; the PN scaffold body was immersed in the mixture to react and obtain polydopamine-modified PN scaffold body; (3) Preparation of DFO@GMs: Gelatin, deferoxamine and water were mixed to obtain solution A; mineral oil and Span-80 were mixed to obtain solution B; solution A, solution B and glutaraldehyde were cross-linked to obtain DFO@GMs; (4) Preparation of DFO@GMs-pDA / PN composite scaffold: DFO@GMs, ethanol and phosphate buffer solution were mixed to obtain a mixture; the polydopamine modified PN scaffold body was immersed in the mixture to react and obtain DFO@GMs-pDA / PN composite scaffold.

2. The method for preparing the DFO@GMs-pDA / PN composite scaffold that promotes vascularization and osteogenic formation according to claim 1, characterized in that, In step (1), the electrospinning solvent includes N,N-dimethylformamide and dichloromethane; the mass ratio of N,N-dimethylformamide and dichloromethane is 3~4:1; the mass ratio of nano-clay, polycaprolactone and electrospinning solvent is 0.12~0.15:1.5~2.5:8~10.

3. The method for preparing the DFO@GMs-pDA / PN composite scaffold that promotes vascularization and osteogenic formation according to claim 2, characterized in that, In step (1), the mixing temperature is 55~65℃, the mixing speed is 100~200r / min, and the mixing time is 50~60min.

4. The method for preparing the DFO@GMs-pDA / PN composite scaffold for promoting vascularization and osteogenic formation according to any one of claims 1 to 3, characterized in that, In step (1), the electrospinning parameters are set as follows: positive voltage is 11.5~12.5kV, negative voltage is 2.3~2.6kV, injection speed is 0.14~0.16mm / min, receiving distance is 8~12cm, receiving speed is 30~35r / min, and receiving time is 2.5~3h.

5. The method for preparing the DFO@GMs-pDA / PN composite scaffold that promotes vascularization and osteogenic formation according to claim 4, characterized in that, In step (2), the mass ratio of dopamine powder to tris(hydroxymethyl)aminomethane in the buffer solution is 0.2:0.11~0.12; the reaction time is 10~14h; and the stirring rate is 200~250r / min.

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

7. The method for preparing the DFO@GMs-pDA / PN composite scaffold that promotes vascularization and osteogenic formation according to claim 6, characterized in that, In step (3), the temperature of the crosslinking reaction is 3~4℃ and the time of the crosslinking reaction is 20~30min.

8. The method for preparing the DFO@GMs-pDA / PN composite scaffold for promoting vascularization and osteogenic formation according to claim 1, 5, 6 or 7, characterized in that, In step (4), the mass-to-volume ratio of DFO@GMs, ethanol and phosphate buffer solution is 200 mg: 47~50 mL: 45~51 mL; the reaction time is 10~14 h; and the stirring rate is 200~250 r / min.

9. The DFO@GMs-pDA / PN composite scaffold prepared by the method of any one of claims 1 to 8 for promoting vascularization and osteogenic formation.

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