4D printing shape memory composite stent for delivering exosomes and magnesium ions and preparation method and application thereof

Abstract

The application discloses a 4D printing shape memory composite stent for delivering exosomes and magnesium ions and a preparation method and application thereof. The preparation method comprises the following steps: (1) mixing hydroxyapatite and silicon dioxide nanoparticles with poly(D, L-lactic acid-co-trimethylene carbonate) in dichloromethane to obtain an oil phase; adding exosomes into a magnesium oxide nanoparticle water dispersion to obtain an aqueous phase; and mixing to obtain a water-in-oil Pickering emulsion; and (2) using a 4D printing technology to prepare the composite porous shape memory stent (PHS / MgO-EXO) loaded with exosomes by using the Pickering emulsion ink. The stent has a multi-stage interconnected pore structure, and can promote cell osteogenic differentiation and bone tissue regeneration by synergistically releasing exosomes and magnesium ions. The stent also has a temperature-sensitive shape memory function, can restore the original shape under a slightly higher body temperature stimulus, can accurately match irregular bone defects, and is suitable for minimally invasive repair treatment of clinical irregular bone defects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and tissue engineering technology, specifically relating to an intelligent bone repair scaffold with shape memory and exosome / magnesium ion synergistic delivery functions, as well as its preparation method and application. Background Technology

[0002] Orthopedic interventions for irregular bone defects caused by trauma, tumors, or infections still present numerous challenges. Currently, the main methods for treating bone defects include autologous bone grafting, allogeneic bone grafting, and the use of bioinert materials. These "gold standard" methods for treating bone defects face several problems, such as limited bone resources, immune rejection, and high postoperative morbidity. Furthermore, surgeons often need to reshape irregular defects into standard geometries, which increases surgical complexity and causes additional tissue damage. Therefore, a shape memory scaffold that can adapt to irregular bone defect sites and form good interfacial integration is of great research value.

[0003] Studies have confirmed that composite scaffolds loaded with stem cells can promote bone formation. However, the clinical application of these stem cell therapies has been hampered by manufacturing and regulatory issues, scalability of cell therapies, immunogenicity, efficacy, safety, and the cost of grafts and constructs. Exosome therapy, as a promising cell-free replacement therapy, consists of cell-derived nanoscale extracellular vesicles (30-150 nm). Rich in bioactive molecules such as proteins, nucleic acids, lipids, and other biomolecules, they can transmit intercellular information and participate in intercellular interactions. The biological function of exosomes in the bone microenvironment is key to paracrine and endocrine signaling. Studies have shown that they can promote bone regeneration. However, exosomes are easily degraded in vivo, requiring carriers for sustained release and targeted delivery. Chinese invention patent CN202410074704.7 describes a process where exosomes are mixed with hydrazide-grafted hyaluronic acid, then mixed with aldehyde-modified hyaluronic acid, and reacted to obtain an exosome-hydrogel system. The hydrogel prepared by this invention for exosome delivery integrates multiple functions, exhibits good biocompatibility, and possesses injectable, self-healing, tissue adhesion, and exosome binding properties. However, the exosome delivery capacity of this hydrogel scaffold is insufficient, making long-term sustained release difficult. Therefore, further development of exosome-loaded scaffolds to facilitate the retention and long-term controlled release of exosomes at the affected site is of positive significance for improving the quality and efficiency of tissue regeneration.

[0004] Intelligent manufacturing of porous scaffolds has seen rapid development in the field of tissue engineering, especially 3D printing technology, which can meet the diverse needs of multi-level porous biological scaffolds in tissue engineering. By using a 3D printer to deposit biomaterials layer by layer into a three-dimensional solid scaffold, the shape, pore size, and interpore connectivity of the biological scaffold can be precisely controlled according to actual needs, meeting the requirements of personalized scaffold manufacturing. Scaffolds prepared by 3D printing Pickering emulsions have multi-scale structures with micron-level macropores and nano-level micropores, mimicking the multi-scale structure of natural bone. Chinese invention patent CN201910132226.X prepares scaffolds using 3D printing of Pickering emulsions containing graphene oxide, which possess photothermal driven shape memory properties and can be deformed by near-infrared light. However, its research focuses only on the deformation mechanism of the scaffold and lacks exploration of its osteogenic function. Another Chinese invention patent, CN201910132509.4, constructs a magnetocalorically responsive shape memory scaffold using 3D printing of iron oxide nanoparticles. Its research focuses on preparing the magnetocalorically responsive scaffold, neglecting its biological applications, biocompatibility, and the utilization of its porous structure. Patent CN109701084A prepares a composite multi-scale porous biological scaffold using 3D printing of a high internal phase emulsion made from bioactive nanoparticles / biodegradable polyester. However, this high internal phase emulsion is unstable and prone to demulsification during printing.

[0005] Existing scaffold materials are multifunctional yet limited, failing to simultaneously meet multiple requirements such as structural adaptation, osteogenic induction, and immune regulation. 4D printing technology introduces a temporal dimension to the static structure of 3D printing, enabling objects to actively change shape under external stimuli (magnetic fields, light, temperature) through smart materials (such as shape memory polymers). This allows them to adapt to complex shape environments (such as precise fitting of irregular bone defects), overcoming the static limitations of traditional scaffolds and providing a new approach to constructing dynamically adaptable smart scaffolds. Furthermore, magnesium ions, as an essential element for the human body, promote osteogenic differentiation and angiogenesis. Therefore, developing a composite scaffold that can synergistically deliver exosomes and magnesium ions and possesses shape memory function has significant clinical implications and application prospects. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention aims to provide a 4D-printable shape memory composite scaffold. It encapsulates exosomes and magnesium oxide using Pickering emulsion, and the printing of the Pickering emulsion constructs a multi-scale porous composite scaffold, achieving synergistic encapsulation and sustained release of exosomes and magnesium ions to promote bone defect repair and realize cell-free therapy. It also possesses minimally invasive implantation capabilities and precise morphological adaptation.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] A method for preparing a 4D-printed shape memory composite scaffold delivering exosomes and magnesium ions involves mixing hydroxyapatite and silica nanoparticles with poly(D,L-lactic acid-co-trimethylene carbonate) (PLMC) in dichloromethane to obtain an oil phase. An aqueous phase is obtained by adding exosomes to an aqueous dispersion of magnesium oxide nanoparticles. A stable water-in-oil Pickering emulsion is then prepared as the printing ink. The composite porous shape memory scaffold (PHS / MgO-EXO) loaded with exosomes (EXO) and magnesium ions is prepared using the Pickering emulsion ink obtained through 4D printing. The magnesium oxide nanoparticles, placed in the aqueous phase, can both release magnesium ions and adsorb exosomes into the emulsion microspheres through positive and negative charge attraction. The composite porous scaffold can release magnesium ions and sustainably release exosomes to promote osteogenic differentiation.

[0009] Specifically, the following steps are included: a) Culture, isolation and identification of exosomes derived from bone marrow mesenchymal stem cells, wherein the exosomes are EXO; b) Preparation of oil phase: PLMC, hydrophobic silica and hydroxyapatite are dispersed in dichloromethane and ultrasonically treated to form a homogeneous oil phase, wherein the hydroxyapatite is modified by hydrophobic polylactic acid with carboxyl-terminated groups. c) Preparation of aqueous phase: Magnesium oxide nanoparticles and exosomes were dispersed in deionized water to obtain an aqueous phase; d) Preparation of Pickering emulsion: The aqueous phase is added to the oil phase and vortex emulsified to form a water-in-oil emulsion; e) 4D printing: The above emulsion is used as a bio-ink for low-temperature 4D printing to construct a scaffold with a pre-defined structure; f) Post-processing: freeze-drying to remove solvent, obtaining the composite scaffold.

[0010] In the technical solution of this invention, the hydroxyapatite is obtained by hydrophobic modification of polylactic acid. The method is to ultrasonically mix hydroxyapatite and polylactic acid in an organic solvent (one of dichloromethane, trichloromethane, xylene, and acetone) at a mass ratio of 1:1 to 1:2 until the solvent evaporates, and then dry it in a vacuum oven at 220-400°C for 24-48 hours to obtain hydrophobic hydroxyapatite.

[0011] In the technical solution of the present invention, the silicon dioxide is hydrophobic silicon dioxide with a diameter of less than 500 nm.

[0012] In the technical solution of this invention, the magnesium oxide is prepared by a co-precipitation method, the specific steps of which are as follows: magnesium nitrate hexahydrate (Mg(NO3)2·6H2O) and sodium hydroxide are dissolved in 100 mL of deionized water at a mass ratio of 3:1 to 3:1.5. The two solutions are mixed and stirred. After stirring at room temperature for 1-5 hours, the mixture is stored at room temperature for aging for 12-48 hours. The obtained precipitate is washed with water and ethanol, and the clean precipitate is dried in an oven at 50-100℃. Then, the dried sample is calcined in a muffle furnace at 400-600℃ for 5-24 hours. The sample is then removed and ground to obtain magnesium oxide nanoparticles.

[0013] In the technical solution of the present invention, the exosomes are EXO, and the source includes at least one of bone marrow mesenchymal stem cells, macrophages, embryonic stem cells, and adipose stem cells.

[0014] In the technical solution of this invention, the exosome extraction includes the following steps: 1) extracting bone marrow mesenchymal stem cells at 1×10⁻⁶ cm⁻¹. 6 Cells were seeded in 75 cm cell culture dishes and cultured. When the cells reached 75% confluence, the medium was replaced with serum-free DMEM and cultured for another 24 hours, after which the medium was collected. 2) The collected medium was subjected to gradient centrifugation: 300×g (10 min), 2,000×g (10 min), and 10,000×g (30 min) in three consecutive steps to remove some cell debris and impurities, and then filtered through a 0.22 μm filter. Subsequently, it was ultracentrifuged at 100,000×g for 70 min to obtain the raw exosome extract, washed once with PBS, and then ultracentrifuged again at 100,000×g for 70 min. The resulting precipitate was the pure exosomes. Finally, the exosomes were resuspended in an appropriate volume of PBS for storage or use. The concentration and total mass of the prepared exosomes were determined using the Micro BCA protein assay kit, qualitative analysis was performed by Western blotting, and a suitable amount of exosome suspension was observed by transmission electron microscopy.

[0015] In the technical solution of the present invention, the 4D printing technology is configured with the following parameters: printing temperature of 0-10℃, printing thickness of 2-20mm, and printing time of 1-5 minutes.

[0016] In the technical solution of this invention, the content of each component of the composite scaffold is as follows: the concentration of hydrophobic hydroxyapatite in the oil phase is 0.1-0.5 g / mL, the concentration of hydrophobic silica is 0.01-0.1 g / mL, and the concentration of PLMC is 0.1-0.5 g / mL. In the technical solution of this invention, the concentration of magnesium oxide in the aqueous phase is 0.5-4 mg / mL, and the concentration of exosomes in the aqueous phase is 0.001-0.1 mg / mL.

[0017] In the technical solution of this invention, the poly(D,L-lactic acid-co-trimethylene carbonate) is a shape-memory biodegradable polymer. Typically, shape-memory biodegradable polymers include polylactic acid. The polymer can be any one of the following biomedical polymer materials: glycolic acid copolymer, polycaprolactone, polyurethane, and other block copolymers. Of course, the polymer is not limited to the polymers listed above, and other shape memory polymers can also be selected according to the needs of actual applications.

[0018] In the technical solution of this invention, the glass transition temperature of the shape memory scaffold is designed to be slightly higher than body temperature (37°C), between 38°C and 45°C, allowing for autonomous control of the scaffold's shape changes. If the temperature triggering shape memory is 37°C, we cannot autonomously control the deformation of the scaffold. During implantation, the scaffold will actively deform, making manual control difficult. A temperature slightly higher than body temperature (41°C) allows for easy control of scaffold deformation simply by heating (e.g., with warm PBS, infrared photothermal therapy, etc.). The scaffold can be reduced in size by changing its initial shape, deforming into any shape, and then quickly returning to its initial shape in a hot water bath, fitting tightly to the bone defect. This temporary shape can return to its initial state, perfectly fitting and supporting the damaged bone defect. This shape memory characteristic minimizes damage to bone tissue during implantation and ensures good fit with the defect site.

[0019] In the technical solution of the present invention, the Pickering emulsion is a water-in-oil emulsion, and magnesium oxide and exosomes are encapsulated in Pickering emulsion microspheres due to the attraction of positive and negative charges.

[0020] In this invention, exosomes and magnesium oxide are encapsulated in Pickering emulsion microspheres, which are then 4D printed and freeze-dried to obtain a shape-memory porous scaffold loaded with exosomes and magnesium oxide. 4D printing provides the scaffold with a millimeter-scale macroporous structure (0.5-2 mm), while the Pickering emulsion, after sublimation, produces a micrometer-scale microporous structure (5-50 μm). In the first stage, exosomes and magnesium ions are released through the porous structure; in the second stage, exosomes and magnesium ions are released as the scaffold degrades. The continuously released exosomes can be phagocytosed by co-cultured cells. The slowly released magnesium ions and exosomes promote cell adhesion, proliferation, and osteogenic differentiation, achieving excellent cell-free therapeutic effects. In-depth mechanistic studies and multi-omics analysis show that the PHS / 1MgO-0.1EXO scaffold regulates osteogenic differentiation of mBMSCs through the Hippo signaling pathway and promotes tissue regeneration through immune regulation mediated by the TNF-α and IL-17 pathways.

[0021] The above method produces a 4D-printed shape memory composite scaffold for delivering exosomes and magnesium ions, which is suitable for minimally invasive repair treatment of irregular bone defects in clinical practice. It can be used to prepare minimally invasive repair treatment materials or drugs for irregular bone defects in clinical practice.

[0022] Compared to CN109701084A, the Pickering emulsion of this invention is a low-internal-phase emulsion. Combined with low-temperature printing and freeze-drying technology, the prepared scaffold exhibits higher pore connectivity, achieving higher porosity and pore connectivity with a smaller inward volume. The low-internal-phase emulsion constructed in this invention stably encapsulates nano-magnesium oxide particles and exosomes in the aqueous phase. The positively charged magnesium oxide adsorbs the negatively charged exosomes, stabilizing the exosomes within the pores and achieving a slow-release effect of exosomes and magnesium ions. The low-temperature printing platform constructed in this invention helps protect the stability of the emulsion and exosomes, making the emulsion less prone to structural damage during printing. Thus, a scaffold with high porosity and higher connectivity can be constructed with a low internal phase.

[0023] The beneficial effects of this invention are as follows: (1) Dual-release porous scaffold: Exosomes and magnesium oxide are encapsulated in Pickering emulsion to achieve synergistic sustained release of exosomes and magnesium ions, thereby enhancing osteogenic induction. The composite scaffold can achieve synergistic sustained release of magnesium ions and exosomes, with a magnesium ion release cycle of no less than 4 weeks and an exosome release cycle of no less than 9 days.

[0024] (2) Precise shape adaptation: The stent achieves minimally invasive implantation and precise shape adaptation by triggering the shape memory effect through body temperature or external temperature. The stent has thermosensitive shape memory properties and can recover its original shape within 5-60 seconds under stimulation at 38℃-45℃, with a shape recovery rate ≥90% and a shape fixation rate ≥95%. It precisely matches irregular bone defects and meets the clinical needs for the repair of irregular bone defects.

[0025] (3) Connected hierarchical porous structure: Combining 4D printing to prepare macroporous structures with emulsion template method to prepare microporous structures is beneficial for cell migration, nutrient delivery and tissue ingrowth.

[0026] (4) Simple preparation process: The process is controllable by combining 4D printing technology with Pickering emulsion template method, which is suitable for personalized customization and large-scale production. Attached Figure Description

[0027] Figure 1a SEM image of MgO; Figure 1b This is a mapping image of MgO; Figure 1c This is a statistical diagram of the particle size distribution of MgO; Figure 1d The XRD pattern of MgO; Figure 1e The Zeta charge diagram for MgO; Figure 2a To observe the morphology of EXO using transmission electron microscopy (TEM); Figure 2b A size distribution chart of EXO; Figure 2c This is the potential diagram of EXO; Figure 2d Western blot characterization of stem cells and EXO; Figure 3a SEM images and EDS elemental composition of scaffolds with different magnesium oxide contents; Figure 3b The graph shows the degradation performance of the stent. Figure 3c pH changes during scaffold degradation; Figure 3d The magnesium ion sustained-release curve of the stent; Figure 4 Differential scanning thermal analysis of the PHS / 1MgO-0.1EXO scaffold; Figure 5a Image of the shape memory effect of the PHS / 1MgO-0.1EXO scaffold; Figure 5b The shape memory recovery rate of the PHS / 1MgO-0.1EXO scaffold after 5 cycles; Figure 5c The shape memory recovery rate and fixation rate of the PHS / 1MgO-0.1EXO scaffold; Figure 6a The growth activity of mBMSCs on scaffolds with different magnesium oxide nanoparticle contents was detected by CCK-8. Figure 6b The study investigated the liveness and cell adhesion of mBMSCs on scaffolds with different magnesium oxide nanoparticle contents. Figure 7a ALP staining of mBMSCs after 7 days of culture on scaffolds with different magnesium oxide nanoparticle contents; Figure 7b The expression of relevant osteogenic genes in mBMSCs after 7 days of culture on scaffolds with different magnesium oxide nanoparticle contents; Figure 8a The exosomes are released cumulatively from different scaffolds; Figure 8b The uptake of exosomes by mBMSCs; Figure 8c The effect of scaffolds with different exosome contents on the promotion of osteogenic-related gene expression; Figure 9a Enrichment of the GO pathway between the PHS / 1MgO-0.1EXO group and the blank control group; Figure 9b Enrichment of the KEGG upregulated pathway between the PHS / 1MgO-0.1EXO group and the blank control group; Figure 10a Micro-CT images of skull defects in rats treated with implanted scaffolds; Figure 10b The BV / TV ratios were calculated at 4 and 8 weeks post-stent implantation. Figure 10c H&E and MTS staining histological evaluation of the skull defect site 8 weeks after stent implantation. Detailed Implementation

[0028] This invention provides a method for preparing a 4D-printed shape memory composite scaffold for delivering exosomes and magnesium ions. The invention will be further described in detail below with reference to the embodiments, but the implementation of the invention is not limited thereto.

[0029] A method for fabricating a 4D-printed shape memory composite scaffold that delivers exosomes and magnesium ions, the scaffold being able to induce a shape memory effect at a temperature slightly above body temperature, is constructed using a combination of Pickering emulsion template method and 4D printing technology, and possesses a multi-scale porous structure. The exosomes are secreted by mouse bone marrow mesenchymal stem cells.

[0030] Example 1 Fabrication of 4D-printed shape memory scaffolds: Preparation of the MgO composite scaffold: First, a water-in-oil composite Pickering emulsion ink was prepared. Simply put, 0.6 g of PLMC was dissolved in 6 mL of dichloromethane (DCM). Then, 0.3 g of hydroxyapatite and 0.12 g of silica were added to the PLMC / DCM solution, and the mixture was sonicated in an ice-water bath for 30 minutes to obtain the oil phase. Deionized water was then added to the oil phase as the aqueous phase, and the mixture was stirred at 3000 rpm using a vortex mixer to form a homogeneous water-in-oil composite Pickering emulsion. The prepared composite Pickering emulsion was transferred to a 50 mL syringe and connected to a V-shaped nozzle (0.4 mm inner diameter). A pre-designed CAD model was then opened in the printing software, and the scaffold was printed layer by layer using a Bio-Printer™ V1.2 printer equipped with a pneumatically driven extrusion syringe. After the model was completed, the scaffold was freeze-dried for 24 hours to remove the solvent. The resulting scaffold was named "PHS".

[0031] Example 2 Fabrication of a 4D-printed shape memory composite scaffold for delivering magnesium ions: Preparation of the MgO composite scaffold: First, a water-in-oil composite Pickering emulsion ink was prepared. Simply put, 0.6 g of PLMC was dissolved in 6 mL of dichloromethane (DCM). Then, 0.3 g of hydroxyapatite and 0.12 g of silica were added to the PLMC / DCM solution, and the mixture was sonicated in an ice-water bath for 30 minutes to obtain the oil phase. MgO nanoparticles were dispersed in deionized water to obtain the aqueous phase (concentration 0.05 mg / mL). The aqueous phase was then added to the oil phase, and the mixture was stirred at 3000 rpm using a vortex mixer to form a homogeneous water-in-oil composite Pickering emulsion. The prepared composite Pickering emulsion was transferred to a 50 mL syringe and connected to a V-shaped nozzle (0.4 mm inner diameter). A pre-designed CAD model was then opened in the printing software, and the scaffold was printed layer by layer using a Bio-Printer™ V1.2 printer equipped with a pneumatically driven extrusion syringe. After the model was completed, the scaffold was freeze-dried for 24 hours to remove the solvent. The resulting stent was named "PHS / 0.05MgO".

[0032] Example 3 Fabrication of a 4D-printed shape memory composite scaffold for delivering magnesium ions: Preparation of the MgO composite scaffold: First, a water-in-oil composite Pickering emulsion ink was prepared. Simply put, 0.6 g of PLMC was dissolved in 6 mL of dichloromethane (DCM). Then, 0.3 g of hydroxyapatite and 0.12 g of silica were added to the PLMC / DCM solution, and the mixture was sonicated in an ice-water bath for 30 minutes to obtain the oil phase. MgO nanoparticles were dispersed in deionized water to obtain the aqueous phase (concentration 1 mg / mL). Then, the aqueous phase was added to the oil phase, and the mixture was stirred at 3000 rpm using a vortex mixer to form a homogeneous water-in-oil composite Pickering emulsion. The prepared composite Pickering emulsion was transferred to a 50 mL syringe and connected to a V-shaped nozzle (0.4 mm inner diameter). A pre-designed CAD model was then opened in the printing software, and the scaffold was printed layer by layer using a Bio-Printer™ V1.2 printer equipped with a pneumatically driven extrusion syringe. After the model was built, the scaffold was freeze-dried for 24 hours to remove the solvent. The resulting scaffold was named "PHS / 1MgO".

[0033] Example 4 Fabrication of a 4D-printed shape memory composite scaffold for delivering magnesium ions: Preparation of the MgO composite scaffold: First, a water-in-oil composite Pickering emulsion ink was prepared. Simply put, 0.6 g of PLMC was dissolved in 6 mL of dichloromethane (DCM). Then, 0.3 g of hydroxyapatite and 0.12 g of silica were added to the PLMC / DCM solution, and the mixture was sonicated in an ice-water bath for 30 minutes to obtain the oil phase. MgO nanoparticles were dispersed in deionized water to obtain the aqueous phase (concentration 2 mg / mL). The aqueous phase was then added to the oil phase, and the mixture was stirred at 3000 rpm using a vortex mixer to form a homogeneous water-in-oil composite Pickering emulsion. The prepared composite Pickering emulsion was transferred to a 50 mL syringe and connected to a V-shaped nozzle (0.4 mm inner diameter). A pre-designed CAD model was then opened in the printing software, and the scaffold was printed layer by layer using a Bio-Printer™ V1.2 printer equipped with a pneumatically driven extrusion syringe. After the model was built, the scaffold was freeze-dried for 24 hours to remove the solvent. The resulting scaffold was named "PHS / 2MgO".

[0034] Example 5 A method for fabricating a 4D-printed shape memory composite scaffold for delivering exosomes and magnesium ions includes the following steps: First, a water-in-oil composite Pickering emulsion ink was prepared. Simply put, 0.6 g of PLMC was dissolved in 6 mL of DCM. Then, 0.3 g of hydroxyapatite and 0.12 g of silica were added to the PLMC / DCM solution, and the mixture was sonicated in an ice-water bath for 30 minutes to obtain the oil phase. MgO nanoparticles were dispersed in deionized water to obtain the aqueous phase (concentration: 1 mg / mL), and exosomes (concentration: 0.025 mg / mL) were added to the aqueous phase. The aqueous phase was then added to the oil phase, and the mixture was stirred at 3000 rpm using a vortex mixer to form a homogeneous water-in-oil composite Pickering emulsion (W / O). The prepared composite Pickering emulsion was transferred to a 50 mL syringe, and a V-shaped nozzle (inner diameter: 0.4 mm) was connected. A pre-designed CAD model was then opened in the printing software, and the support was printed layer by layer using a Bio-Printer™ V1.2 printer equipped with a pneumatically driven extrusion syringe. After the model was built, the support was freeze-dried for 24 hours to remove the solvent. The resulting stent was named "PHS / 1MgO-0.025EXO".

[0035] Example 6 A method for fabricating a 4D-printed shape memory composite scaffold for delivering exosomes and magnesium ions includes the following steps: First, a water-in-oil composite Pickering emulsion ink was prepared. Simply put, 0.6 g of PLMC was dissolved in 6 mL of DCM. Then, 0.3 g of hydroxyapatite and 0.12 g of silica were added to the PLMC / DCM solution, and the mixture was sonicated in an ice-water bath for 30 minutes to obtain the oil phase. MgO nanoparticles were dispersed in deionized water to obtain the aqueous phase (concentration: 1 mg / mL), and exosomes (concentration: 0.05 mg / mL) were added to the aqueous phase. The aqueous phase was then added to the oil phase, and the mixture was stirred at 3000 rpm using a vortex mixer to form a homogeneous water-in-oil composite Pickering emulsion (W / O). The prepared composite Pickering emulsion was transferred to a 50 mL syringe, and a V-shaped nozzle (inner diameter: 0.4 mm) was connected. A pre-designed CAD model was then opened in the printing software, and the support was printed layer by layer using a Bio-Printer™ V1.2 printer equipped with a pneumatically driven extrusion syringe. After the model was built, the support was freeze-dried for 24 hours to remove the solvent. The resulting stent was named "PHS / 1MgO-0.05EXO".

[0036] Example 7 A method for fabricating a 4D-printed shape memory composite scaffold for delivering exosomes and magnesium ions includes the following steps: First, a water-in-oil composite Pickering emulsion ink was prepared. Simply put, 0.6 g of PLMC was dissolved in 6 mL of DCM. Then, 0.3 g of hydroxyapatite and 0.12 g of silica were added to the PLMC / DCM solution, and the mixture was sonicated in an ice-water bath for 30 minutes to obtain the oil phase. MgO nanoparticles were dispersed in deionized water to obtain the aqueous phase (concentration: 1 mg / mL), and exosomes (concentration: 0.1 mg / mL) were added to the aqueous phase. The aqueous phase was then added to the oil phase, and the mixture was stirred at 3000 rpm using a vortex mixer to form a homogeneous water-in-oil composite Pickering emulsion (W / O). The prepared composite Pickering emulsion was transferred to a 50 mL syringe, and a V-shaped nozzle (inner diameter: 0.4 mm) was connected. A pre-designed CAD model was then opened in the printing software, and the support was printed layer by layer using a Bio-Printer™ V1.2 printer equipped with a pneumatically driven extrusion syringe. After the model was built, the support was freeze-dried for 24 hours to remove the solvent. The resulting stent was named "PHS / 1MgO-0.1EXO".

[0037] A 4D-printed shape memory composite scaffold for delivering exosomes and magnesium ions for bone defect repair.

[0038] Experimental section: 1. Characterization of magnesium oxide nanoparticles: like Figures 1a to 1eAs shown, the microstructure and particle size of MgO can be observed using scanning electron microscopy (SEM). MgO exhibits a granular morphology with an average particle size of approximately 40 nm and relatively uniform particle size. EDS images show that Mg and O elements are uniformly distributed on the MgO nanoparticles. XRD was used to characterize its structure, revealing relatively complete crystal planes (111), (200), (220), (311), and (222), with sharp crystal shapes, indicating high crystallinity of the prepared MgO nanoparticles. Therefore, the MgO nanoparticles prepared by the co-precipitation synthesis method possess good crystal form, high crystallinity, and small grain size. Furthermore, the zeta potential of the MgO nanoparticles is positive 19. Since exosomes are negatively charged, the introduction of positively charged MgO nanoparticles into the aqueous phase in this study allows for effective adsorption and stabilization of exosomes within the Pickering emulsion microspheres through electrostatic interactions, potentially achieving good controlled release.

[0039] 2. Characterization of exosomes: see Figures 2a to 2d The morphology of exosomes was observed and recorded using transmission electron microscopy (TEM). First, 10 μL of the obtained exosomes were added to a copper mesh to form water droplets, and after absorbing excess liquid, they were dried. For staining, 10 μL of 2% phosphotungstic acid was added, and staining was performed in the dark for 2-5 min. After drying, the residues were used for TEM observation. Vesicles with a diameter of 50-150 nm, arranged in a disc-like, teacup-like shape, indicated the presence of exosomes. Exosome particle size detection: Nanoparticle tracking analysis (NTA) was used to determine the sample concentration and particle size distribution. NTA data showed a single, distinct main peak at 92 nm, indicating that the prepared exosomes were uniform in size and exhibited the particle size characteristics of composite exosomes. The charge of the exosomes was determined using a Zetasizer Nanometer. In short, 1 mL of exosome suspension was carefully transferred to a syringe, filtered through a 0.22 μm sterile filter, and 1 mL of exosome solution was extracted for Zeta potential detection; the exosomes showed a negative charge. Western blotting was used to identify the exosomes: after centrifugation, the exosomes were lysed using RIPA containing a protease inhibitor. Control group mBMSCs samples were lysed on ice for ten minutes and then centrifuged. The supernatant was used as the lysis product for loading. Following the BCA kit instructions, the protein concentrations of the two groups of samples were obtained. The protein concentrations of the two groups of samples were adjusted to be consistent using lysis buffer. Then, the protein samples were mixed with Western blotting (WB) loading buffer at a ratio of 3:1 to obtain the WB loading samples, which were then subjected to Western blotting (WB) analysis. WB analysis confirmed that mBMSC-EXO highly expressed the exosome protein markers TSG101 and CD63, but did not express the exosome-negative protein marker Calnexin. These results demonstrate that mBMSCs-derived exosomes were successfully extracted.

[0040] 3. Physicochemical property characterization of the stent: see Figure 3aand Figure 3b SEM results showed a uniform, interconnected pore structure, with micropores ranging in size from 5 μm to 50 μm within the composite scaffold framework. As the content of MgO nanoparticles in the aqueous phase increased, the number of interconnected micropores within the composite scaffold framework also increased, and corresponding EDS analysis showed an increase in Mg content. The presence of these interconnected micropores provides a favorable foundation for cell survival, migration, information exchange, and material exchange. More importantly, the interconnected porous structure provides excellent conditions for the delivery of exosomes and magnesium ions. The degradation behavior of the scaffold was tested according to the national standard GB / T 16886.13-2017. The specific steps were as follows: The composite scaffold was immersed in PBS (37℃, 60 rpm) at a mass-to-volume ratio of 0.1 g / mL for 4 weeks. Degradation solution was collected at each time point, and fresh PBS was exchanged. The pH of the solution was measured using a pH meter (Sartorius, PB-10). After 1, 2, 3, and 4 weeks of culture, the scaffold samples were removed and rinsed several times with deionized water to remove precipitated salts. The rinsed scaffolds were then freeze-dried for 48 hours, and the remaining weight was measured using a digital balance. With increasing time, the weight loss of each group of scaffolds continuously increased, with the rate of weight loss increasing sequentially due to the increasing MgO nanoparticle content. It is inferred that the weight reduction of the scaffolds is due to continuous degradation with increasing immersion time, primarily through the slow release and decomposition of MgO nanoparticles into magnesium ions, and the degradation of the matrix polymer PLMC constituting the scaffold. Furthermore, a good bio-scaffold should possess degradation products with good biocompatibility with surrounding tissues and cells, and should not induce inflammatory or foreign body reactions. For example, the degradation process should maintain a relatively stable pH level within the tissue. Figure 3c It can be seen that the pH value did not change much during the degradation of the composite scaffold, remaining between approximately 7.2 and 7.4. This is because the MgO nanoparticles combine with water to produce slightly alkaline OH-. - The MgO nanoparticles neutralize some of the weakly acidic substances produced during the degradation of the polymer PLMC, bringing the overall pH closer to neutral. Therefore, the composite scaffold constructed from MgO nanoparticles can regulate the acid-base balance of the scaffold during the degradation process, potentially improving the biocompatibility of the biological scaffold.

[0041] The scaffolds were subjected to sustained-release experiments, and the specific steps were as follows: MgO composite scaffolds with different MgO nanoparticle contents were placed in fragrance bottles, and 10 mL of PBS was added. The bottles were then placed on a shaker at 37°C and a shaking speed of 60 rpm for sustained-release testing. Samples were taken at weeks 1, 2, 3, and 4 to test the magnesium ion concentration in the PBS. Figure 3d It can be seen that an MgO composite scaffold was prepared and the slow release of magnesium ions was achieved. The higher the content of MgO nanoparticles, the faster the release.

[0042] See Figure 4 Differential scanning thermal analysis showed that the glass transition temperature of the PHS / 1MgO-0.1EXO scaffold was 41℃.

[0043] 4. Shape memory characterization: The PHS / 1MgO-0.1EXO scaffold was placed in water at 41°C to evaluate its ability to recover its original shape. See the procedure below. Figure 5a Cylindrical and square scaffolds were placed in hot water to bend and fold into smaller shapes, then fixed in cold water to a temporary shape. Upon re-immersion in hot water, the scaffolds were able to return to their original shape, mimicking the small size of clinical bone defect implants. The shape memory performance of the PHS / 1MgO-0.1EXO scaffolds was evaluated using a recovery test. First, the initial height (h0) of a cylindrical scaffold with a diameter of 20 mm was measured. Then, the scaffold was compressed and immersed in warm water at 41°C for 60 seconds to achieve deformation, and the height after deformation (h0) was recorded. d The temporary shape of the support was fixed by immersing it in ice water, thus obtaining the fixed height (h). f Finally, the support was placed back into 41°C water for 60 seconds to achieve final recovery, and the height after recovery (h) was measured. r Shape memory performance is quantified using the following two formulas: Recovery rate (%) = 100 (h) r -h d ) / (h0-h d ); Fixation rate (%) = 100 h f / h d The results are shown below. Figures 5b to 5c The PHS / MgO-0.1EXO scaffold can shrink in size by changing its initial shape and then rapidly return to its original shape in a hot water bath. This temporary shape recovery perfectly conforms to and supports the damaged bone defect. This shape memory property minimizes damage to bone tissue during implantation and ensures good fit to the defect site. Cyclic shape memory evaluation showed consistent performance throughout five test cycles. The scaffold exhibits excellent shape retention rate (R0.1) calculated according to the formula. f = 99.29±0.002%) and recovery rate (R r = 97.14±0.02%, indicating that it possesses clinically applicable shape memory stability suitable for bone regeneration applications. In clinical practice, when dealing with irregular bone defects, it is no longer necessary to cut them into regular shapes. Personalized irregular shape memory scaffolds can be printed. These scaffolds can be made in small sizes to accommodate minimally invasive implantation. Then, by applying a temperature slightly above body temperature, the shape of the scaffold is triggered to recover, thereby precisely conforming to the irregular bone defect.

[0044] 5. See Figures 6a to 6bThe proliferation of mBMSCs on different composite scaffolds was evaluated using the CCK-8 assay. All scaffolds promoted mBMSC proliferation to varying degrees. Simultaneously, live / dead staining observation showed that the scaffolds did not produce significant cytotoxicity, with only a small number of dead cells detected on the surface. Furthermore, FITC and DAPI staining revealed that mBMSCs cells thoroughly covered the scaffold surface. The cells exhibited a spindle-shaped morphology with prominent pseudopodia, and intercellular filaments were also observed, demonstrating the good biocompatibility of the scaffolds prepared in this invention. Excessive magnesium oxide content can cause cytotoxicity; therefore, the PHS / 1MgO scaffold group was selected for subsequent experiments.

[0045] 6. Osteogenic differentiation experiment: see Figures 7a to 7b The effects of ALP staining on osteogenic differentiation of mBMSCs on MgO composite scaffolds of different compositions were investigated; the higher the MgO content, the deeper the staining. Simultaneously, PCR experiments were performed at the molecular level to investigate the expression of osteogenic genes such as ALP, CoL-I, RUNX-2, and OCN. The scaffold significantly upregulated the expression of these osteogenic genes, and the slowly released magnesium ions from the scaffold promoted osteogenic differentiation of mBMSCs. An appropriate magnesium ion content is beneficial for osteogenic differentiation, and the PHS / 1MgO scaffold group was selected for further research.

[0046] After loading exosomes, the effects of scaffold sustained-release of exosomes and promotion of osteogenic differentiation were investigated. (See...) Figures 8a to 8c The cumulative release of exosomes was measured using a BCA protein assay kit, showing that the exosome-loaded scaffold continuously and slowly released exosomes for up to 9 days. These results indicate that the exosome-loaded scaffold is suitable for stable and sustained exosome release. Uptake experiments showed that exosomes could be taken up and internalized by mBMSCs, enabling them to transmit information to the cells. The expression of ALP, CoL-I, RUNX-2, and OCN genes was upregulated, with the upregulation becoming more significant with increasing exosome load. The PHS / 1MgO-0.1EXO scaffold combination showed the best effect in promoting osteodifferentiation.

[0047] See Figure 9a Through gene ontology (GO) analysis, the significance of differentially expressed genes in three aspects—biological processes (BP), cellular components (CC), and molecular functions (MF)—was determined. GO database analysis showed that these significantly upregulated genes could be divided into five categories: cell adhesion and matrix remodeling, regulation of immune-inflammatory homeostasis, epigenetics and metabolic reprogramming, osteogenic differentiation and development. Figure 9bMulti-omics analysis showed that the PHS / 1MgO-0.1EXO scaffold regulates osteogenic differentiation of bone marrow mesenchymal stem cells through the Hippo signaling pathway and promotes tissue regeneration by mediating immune regulation through the TNF-α and IL-17 pathways.

[0048] 7. In vivo bone regeneration effect: See Figure 10. The exosome-loaded composite scaffold PHS / 1MgO-0.1EXO was used to promote bone regeneration in rat skulls. A rat skull defect model was established, and PHS / 1MgO and PHS / 1MgO-0.1EXO scaffolds were implanted into the defect site for treatment. We observed new bone formation in rat skull defects at 4 and 8 weeks after scaffold implantation. Micro-CT scans of the skull after the experiment are shown below. Figure 10a As shown, new bone formation was observed in the 5 mm defect. In the PHS / 1MgO group and the PHS / 1MgO-0.1EXO group, bone formation was relatively small at week 4, but significantly more bone formation was observed at week 8. The healing of the bone defect began in the peripheral region and extended towards the central region. Figure 10b The BV / TV values ​​of the Blank group, PHS / 1MgO group, and PHS / 1MgO-0.1EXO group were 3.1%, 2.9%, and 3.2% at week 4, respectively; and reached 3.1%, 6.2%, and 8.7% at week 8, respectively. The Blank group showed almost no change, while the BV / TV values ​​of the PHS / 1MgO group and the PHS / 1MgO-0.1EXO group increased by two times. These data indicate that the PHS / 1MgO-0.1EXO scaffold has strong potential in promoting new bone growth, consistent with the results of in vitro molecular experiments. To observe bone regeneration in the defect area, samples were sectioned, and H&E and MTS staining of skull tissue was used to evaluate bone formation. (See attached figures). Figure 10c H&E staining showed that the defect areas in the PHS / 1MgO and PHS / 1MgO-0.1EXO groups were covered by bone collagen and new bone tissue, while the Blank group mainly consisted of fibroblasts and proliferating fibrous tissue. Compared with the PHS / 1MgO group, the PHS / 1MgO-0.1EXO group showed a significant increase in new bone formation, while the blank group showed the least amount of new bone formation. Furthermore, MTS staining showed that compared with other groups, the PHS / 1MgO and PHS / 1MgO-0.1EXO groups contained more mature collagen fibers, with the PHS / 1MgO-0.1EXO group having the most. These results indicate that the PHS / 1MgO-0.1EXO scaffold can promote bone regeneration.

[0049] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a 4D-printed shape memory composite scaffold for delivering exosomes and magnesium ions, characterized in that, The oil phase was obtained by mixing hydroxyapatite and silica nanoparticles with poly(D,L-lactic acid-co-trimethylene carbonate) in dichloromethane. The aqueous phase was obtained by adding exosomes to an aqueous dispersion of magnesium oxide nanoparticles. The mixture was then mixed to obtain a stable water-in-oil Pickering emulsion as a printing ink. A composite porous shape memory scaffold loaded with exosomes and magnesium ions was prepared from the 4D printing Pickering emulsion ink.

2. The preparation method according to claim 1, characterized in that, Specifically, the following steps are included: a) Culture, isolation, and identification of exosomes derived from bone marrow mesenchymal stem cells; b) Preparation of oil phase: PLMC, hydrophobic silica and hydroxyapatite are dispersed in dichloromethane and ultrasonically treated to form a homogeneous oil phase, wherein the hydroxyapatite is modified by hydrophobic polylactic acid with carboxyl-terminated groups. c) Preparation of aqueous phase: Magnesium oxide nanoparticles and exosomes were dispersed in deionized water to obtain an aqueous phase; d) Preparation of Pickering emulsion: The aqueous phase is added to the oil phase and vortex emulsified to form a water-in-oil emulsion; e) 4D printing: The above emulsion is used as a bio-ink for low-temperature 4D printing to construct a scaffold with a pre-defined structure; f) Post-processing: freeze-drying to remove solvent, obtaining the composite scaffold.

3. The preparation method according to claim 1, characterized in that, The hydroxyapatite is obtained by hydrophobic modification of polylactic acid. The method is to ultrasonically mix hydroxyapatite and polylactic acid in an organic solvent at a mass ratio of 1:1 to 1:2 until the solvent evaporates, and then dry it in a vacuum oven at 220-400℃ for 24-48 hours to obtain hydrophobic hydroxyapatite.

4. The preparation method according to claim 1, characterized in that, The magnesium oxide was prepared by a co-precipitation method, the specific steps of which are as follows: magnesium nitrate hexahydrate (Mg(NO3)2·6H2O) and sodium hydroxide were dissolved in deionized water at a mass ratio of 3:1 to 3:1.

5. The two solutions were mixed and stirred. After stirring at room temperature for 1-5 hours, the mixture was stored at room temperature for aging for 12-48 hours. The precipitate was washed with water and ethanol. The clean precipitate was placed in an oven at 100-200℃ to dry the moisture. Then the dried sample was placed in a muffle furnace at 400-600℃ for calcination for 5-24 hours. The sample was then removed and ground to obtain magnesium oxide nanoparticles.

5. The preparation method according to claim 1, characterized in that, The concentration of magnesium oxide in the aqueous phase is 0.5-4 mg / mL.

6. The preparation method according to claim 1, characterized in that, The exosomes are EXO, and their sources include at least one of bone marrow mesenchymal stem cells, macrophages, embryonic stem cells, and adipose stem cells; the concentration of exosomes in the aqueous phase is 0.001-0.1 mg / mL.

7. The preparation method according to claim 1, characterized in that, The parameters set for the 4D printing technology include: printing temperature of 0-10℃, printing thickness of 2-20mm, and printing time of 1-5 minutes.

8. The 4D-printed shape memory composite scaffold for delivering exosomes and magnesium ions obtained by the preparation method according to any one of claims 1-7, characterized in that, The composition of the composite scaffold is as follows: the concentration of hydrophobic hydroxyapatite in the oil phase is 0.1-0.5 g / mL, the concentration of hydrophobic silica is 0.01-0.1 g / mL, and the concentration of PLMC is 0.1-0.5 g / mL.

9. The 4D-printed shape memory composite scaffold for delivering exosomes and magnesium ions obtained by the preparation method according to any one of claims 1-7, characterized in that, The composite scaffold has a temperature-sensitive shape memory function, and can recover its original shape within 5-60 seconds under stimulation at 38-45℃, with a shape recovery rate of ≥90% and a shape fixation rate of ≥95%.

10. The use of the 4D-printed shape memory composite scaffold for delivering exosomes and magnesium ions as described in claim 8 or 9 in the preparation of minimally invasive repair treatment materials or drugs for clinical irregular bone defects.

Images