Porous microsphere system for bone regeneration and preparation and application thereof

Abstract

The invention discloses a porous microsphere system for bone regeneration and preparation and application thereof, and relates to the technical field of biomedical engineering and tissue engineering.The porous microsphere system is characterized in that a porous polylactic acid-glycolic acid copolymer microparticle library with adjustable pore diameters is prepared by combining a one-step emulsification method with a chemical etching process; by using the porous particles with multiple functions and drug-loading active compounds, the material can simultaneously bear the functions of an ECM simulation stent assembly and a self-assembly bone-like micro-tissue delivery carrier; in addition, the particle can also be used as a multifunctional carrier of various bone repair factors, including bone morphogenetic protein (BMP-2), nano-hydroxyapatite (nHA) and human umbilical vein endothelial cells (HUVECs). Precise regulation and control of the pore size of the microspheres are achieved through a one-step emulsification method and a chemical etching process, the microspheres can efficiently load BMP-2, nHA and HUVECs, multiple requirements of bone regeneration are met, and the formed three-dimensional micro-tissue has osteogenic differentiation and vascularization functions and is suitable for in-vitro models and clinical treatment.

Description

Technical Field

[0001] This invention relates to the fields of biomedical engineering and tissue engineering technology, specifically to a porous microsphere system for bone regeneration and its preparation and application. Background Technology

[0002] Bone tissue regeneration is a crucial topic in clinical medicine, particularly in the repair of bone defects and the treatment of bone diseases. Traditional bone repair materials suffer from limitations such as donor availability and immune rejection. In recent years, extracellular matrix (ECM)-based biomaterials, due to their high specific surface area, have played a vital role as microcarriers in delivery systems, becoming a research hotspot in bone tissue engineering. Co-assembling cells with micron-scale ECM-based biomaterials is a promising strategy for constructing three-dimensional microtissues.

[0003] Existing materials, such as CN110522946B, describe rhBMP-2-loaded bone repair microspheres and their preparation method, belonging to the category of bone repair materials. These microspheres include rhBMP-2 (recombinant bone morphogenetic protein-2) and poly(lactic-coated glycolide) (PLGA) microspheres, with rhBMP-2 encapsulated within the PLGA microspheres. The surface of the microspheres is modified with mussel adhesive protein. However, existing porous microspheres have limited pore size adjustment ranges and single functions, making it difficult to simultaneously meet the multiple needs of osteogenic differentiation, vascularization, and cell delivery in bone regeneration. Furthermore, the preparation processes of existing microspheres are complex, hindering precise pore size control and large-scale production. Therefore, developing a porous microsphere system with adjustable pore size, multifunctional loading, and the ability to guide bone tissue regeneration has significant scientific and application value.

[0004] In view of this, in-depth research was conducted on the above issues, which led to the creation of this case. Summary of the Invention

[0005] The purpose of this invention is to provide a porous microsphere system for bone regeneration, its preparation and application, in order to solve the problems of limited pore size adjustment range and single function of porous microspheres mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing porous PLGA microspheres with adjustable pore size, comprising the following steps: PLGA and porogen PVP were mixed at a weight ratio of 40 / 60 to 100 / 0 and dissolved in dichloromethane to form an oil phase, with a final polymer concentration of 200 g / L. The oil phase is injected into an aqueous phase containing 0.5% PVA, with an oil-to-water volume ratio of 1% to 4%, and emulsified at 10,000–13,000 rpm for 1 minute to form an oil-in-water emulsion. After evaporating dichloromethane at room temperature, the microspheres were freeze-dried to remove residual porogens, thus obtaining the initial porous PLGA microspheres. The initial microspheres were treated with an etching solution containing 0.25 M sodium hydroxide and ethanol at a ratio of 30 / 70 v / v for 30 seconds to 5 minutes to adjust the pore size of the microspheres. Microspheres were collected by filtration, and their pore size and morphology were verified by SEM and ImageJ.

[0007] Preferably, the PLGA / PVP weight ratio is 50 / 50 to 70 / 30, and the oil-water volume ratio is 2%.

[0008] A porous PLGA microsphere loaded with bone repair factors is provided by treating the surface of the microsphere with oxygen plasma to enhance loading efficiency, loading bone morphogenetic protein 2 (BMP-2) by electrostatic adsorption, or loading nano-hydroxyapatite (nHA) through polylysine (PLL) mediated loading.

[0009] Preferably, the method for preparing the microspheres loaded with bone morphogenetic protein-2 (BMP-2) is as follows: the microspheres treated with oxygen plasma are immersed in a solution containing 1 μg / mL BMP-2, 2.5% glycine and 5 mM NaCl, and incubated overnight at room temperature.

[0010] Preferably, the method for preparing the microspheres loaded with nano-hydroxyapatite (nHA) includes: Poly-L-lysine (PLL) and nHA were mixed and left to stand overnight at 60°C. The microspheres treated with oxygen plasma were immersed in a PLL-nHA mixed solution and stirred overnight at 50°C. SEM-EDS analysis showed that the Ca / P elements were evenly distributed.

[0011] Preferably, the microspheres also include loads of human umbilical vein endothelial cells (HUVECs), with each microsphere capable of loading 5–15 HUVECs.

[0012] Preferably, the microsphere loading method for the human umbilical vein endothelial cells (HUVECs) is as follows: the microspheres and HUVECs are co-cultured in a low-absorption plate at a ratio of 1:10-20 for 24 hours, and the mixing ratio is adjusted as needed.

[0013] A method for constructing three-dimensional microtissues for bone regeneration, comprising: The PLGA microspheres loaded with BMP-2, nHA, or HUVECs were co-seeded with human bone marrow mesenchymal stem cells (HMSCs) in a low-adsorption microarray plate at a microsphere:cell ratio of 1:200. Cultured in osteogenic induction medium for 7–14 days to form mixed microtissues; The expression levels of ALP and OPN in the micro-tissues were significantly increased, or CD31 was formed. + Endothelial network.

[0014] Preferably, the three-dimensional microstructure is used for any of the following purposes: Construction of in vitro bone models; Cell therapy for bone defect repair; Regeneration of vascularized bone tissue.

[0015] A porous microsphere system for bone regeneration includes: The above method can be used to prepare PLGA microspheres with adjustable pore size. Functionalized microspheres loaded with BMP-2, nHA, or HUVECs as described above; Three-dimensional bone microtissues formed by co-assembly with HMSCs.

[0016] Compared with the prior art, the beneficial effects of the present invention are: the porous microsphere system for bone regeneration and its preparation and application achieve precise control of microsphere pore size through one-step emulsification and chemical etching processes. The microspheres can efficiently load BMP-2, nHA and HUVECs to meet multiple needs of bone regeneration. The resulting three-dimensional microtissue has both osteogenic differentiation and vascularization functions, and is suitable for in vitro models and clinical treatment.

[0017] Specifically, this invention proposes the use of multifunctional porous microparticles (i.e., microshuttles) loaded with drug-active compounds. This material can simultaneously function as an ECM-mimicking scaffold component and a self-assembling bone-like microtissue delivery carrier. A one-step emulsification method combined with a chemical etching process was developed to prepare a library of porous polylactic-co-glycolic acid (PLGA) microparticles with tunable pore sizes. When co-seeded with human bone marrow mesenchymal stromal cells (HMSCs) in micropores, these microparticles guide cell assembly to form hybrid cell-biomaterial microtissues. Furthermore, these microparticles can also serve as multifunctional carriers of various bone repair factors, including bone morphogenetic protein 2 (BMP-2), nano-hydroxyapatite (nHA), and human umbilical vein endothelial cells (HUVECs). Compared to microtissues containing only HMSCs, the hybrid microtissues containing BMP-2 and nHA-loaded PLGA microparticles showed significantly enhanced osteogenic protein expression; while the hybrid microtissues containing HUVECs-loaded PLGA microparticles exhibited a more complete endothelial network formation capacity. These findings highlight the ability of porous PLGA microcarriers in engineered human bone microtissues for use in in vitro bone model construction and bone regeneration and repair. Attached Figure Description

[0018] Figure 1The diagram shows the preparation process of porous PLGA microspheres and the morphology comparison under different PLGA / PVP ratios (the corresponding microsphere preparation methods in the figure show the SEM images and particle size distribution of microspheres with different PLGA / PVP ratios from 70 / 30 to 40 / 60 w / w and an oil phase / water phase ratio of 2%). Figure 2 A schematic diagram of the preparation of porous PLGA microspheres on a large scale and the verification of their particle size uniformity (the figure shows the macroscopic and microscopic morphology of microspheres obtained by fixing the oil phase / water phase ratio at 2% under different PLGA / PVP ratios in the large-scale preparation of microspheres). Figure 3 A schematic diagram of the multi-parameter optimization results for controlling the pore size of microspheres by chemical etching (the figure shows SEM images of microspheres obtained under different treatment methods: for example, 80 / 20-1%-30s means that the mass ratio of PLGA / PVP is 80 / 20, the volume ratio of oil phase (PLGA / PVP dissolved in dichloromethane) / water phase (PVA polyvinyl alcohol aqueous solution) is 1%, and 30s is the soaking time in NaOH / EtOH mixed solution). Figure 4 Schematic diagram of porous microspheres under extended preparation parameters (microspheres with other pore sizes can be generated by this method, such as the morphology characterization of microspheres with an oil-to-water ratio of 2.8% v / v and PLGA / PVP 80 / 20 w / w). Figure 5 A schematic diagram of the construction and structural characteristics of porous PLGA microsphere-HMSC hybrid microtissue (5 types of microspheres (A) were selected and mixed with HMSCs in micropores to form bone microtissue / assemblies (B), in which porous microspheres simulated the bone matrix, and different microspheres could regulate the distribution of cells in bone microtissue (C)). Figure 6 A schematic diagram illustrating the functional verification of BMP-2-loaded microspheres inducing bone differentiation (PLGA porous microspheres and BMP-2 are electrostatically adsorbed, allowing BMP-2 to be loaded into the microspheres and inducing the expression of bone differentiation markers ALP and OPN in bone microtissues). Figure 7 The schematic diagram of the characterization of the nHA-PLL-PLGA multilayer loading structure (the microspheres were successfully loaded with nHA through PLL (polylysine) and Lewis acid-base reaction, which is actually a PLGA-PLL-nHA three-layer structure. The structure of SEM and EDS mapping can be seen (B), and the elemental analysis of EDS shows the presence of Ca and P elements (C)). Figure 8 A schematic diagram illustrating how nHA-loaded microspheres promote the expression of osteogenic markers in bone microtissue (nHA-loaded microspheres can form bone microtissue with HMSCs and promote the expression of markers ALP and OPN; the figure shows a quantitative comparison of ALP / OPN fluorescence intensity). Figure 9 This is a schematic diagram of the formation of the endothelial network in vascularized bone microtissue (microspheres can simultaneously generate vascularized bone microtissue with HMSCs and HUVECs; the figure shows the CD31-positive tubular structure of the microsphere-HUVEC-HMSC co-culture system). Figure 10 A schematic diagram for quantitative verification of the cell loading capacity of a single microsphere (a single microsphere can load cells, and a macroporous microsphere can load 15 HUVECs. This method can be expanded to load all cells, greatly improving cell load. The figure shows the attachment and distribution of HUVECs on the surface of the microsphere and the loading capacity of 15 cells / sphere). Figure 11 This is a schematic diagram illustrating the protective effect of microspheres on HUVECs in the bone microenvironment (microspheres can protect the survival of HUVECs under bone culture conditions; CD31 in the microsphere-free group is visible). + The HUVECs disappeared, and this method can be extended to the protection and delivery of microspheres to all other cells, and to the loading and protection of all similar porous microspheres. The figure shows CD31. + (Comparison of cell viability and schematic diagram of cell protection mechanism); Figure 12 This is a schematic diagram of the microsphere delivery and bone microtissue system of the present invention. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Please see Figure 1-12 The present invention provides the following technical solutions: Example 1: Preparation of porous PLGA microspheres: Porous PLGA microspheres were prepared using a one-step oil-in-water emulsion method. PLGA (lactide / glycolic acid ratio: 85 / 15, intrinsic viscosity: 0.6 dl / g, Polysciences Inc.) and PVP (K12, molecular weight: ~3500 Da, VWR), used as both the substrate and pore-forming agent, were mixed at a PLGA / PVP weight ratio of 80 / 20 (w / w) and dissolved in dichloromethane (Sigma-Aldrich) to achieve a final polymer concentration of 200 g / L. Different volumes of this polymer solution (i.e., the oil phase) were injected into 30 mL of 0.5% (w / v) PVA solution (i.e., the aqueous phase, Mowiol 8-88, molecular weight: ~67000 Da, degree of hydrolysis 86.7-88.7 mol%, Sigma-Aldrich) to obtain emulsions with oil-water volume ratios of 1%, 1.5%, 2%, 2.5%, 2.8%, 3%, and 4%, respectively. Emulsification was performed for 1 minute at 10,000–13,000 rpm using a high-speed homogenizer (FSH-2a, Vevor) to form oil-in-water droplets. To finely control the pore size of the microspheres, the oil-water volume ratio was kept constant at 2%, and different PLGA / PVP weight ratios were used (70 / 30, 60 / 40, 50 / 50, 40 / 60 w / w). After allowing the dichloromethane in the oil phase to evaporate overnight at room temperature (RT), the emulsion droplets were freeze-dried (FreeZone 2.5 freeze-drying system, Labconco) for two days, resuspended in deionized (DI) water, and centrifuged at 2000 rpm for 5 minutes in 50 mL centrifuge tubes (Eppendorf) to remove residual porogens. The resulting porous microspheres were filtered using stacked cell filters with pore sizes of 100 µm and 40 µm (Corning).

[0021] Example 2: Hole Modification To modify the pore size and porous microstructure of the microspheres, PLGA microspheres with a PLGA / PVP weight ratio of 80 / 20 w / w and an oil-water content of 1% v / v, as well as microspheres with PLGA / PVP weight ratios of 70 / 30, 60 / 40, and 40 / 60 w / w and an oil-water content of 2% v / v, were treated with the etching solution for different times (30 seconds, and 1, 2, 3, and 5 minutes). The etching solution containing the microspheres was washed with deionized (DI) water, and then the microspheres were collected through a 40 µm cellular filter.

[0022] Example 3: Microsphere Characterization The chemical composition of the microspheres was determined using Fourier transform infrared spectroscopy (FTIR, Nicolet iS50, Thermo Fisher Scientific). To observe their morphology and porous structure, the microspheres were first fixed to aluminum posts with conductive carbon tape (Electron Microscopy Sciences, Hatfield) and then sputtered with gold (SC7620, Quorum Technologies) to enhance surface conductivity. The morphology of the microspheres was observed using scanning electron microscopy (SEM, JSM-IT200 InTouchScope, JEOL Ltd.) at 10–15 keV. The particle size distribution and pore size of the microspheres were quantified using ImageJ 1.54f analysis software. The particle size distribution of three hundred microspheres from three independent SEM images was determined. Pore size quantification was performed on five groups of microspheres with different pore sizes (from non-porous to microporous) and similar average diameters (i.e., microspheres I–V).

[0023] Example 4: Preparation of BMP-2-loaded microspheres BMP-2 was selected as a macromolecular biological factor to induce osteogenic differentiation of human bone marrow mesenchymal stem cells (HMSCs). To obtain BMP-2-loaded microspheres, recombinant BMP-2 (Pepro Tech) was first dissolved in a mixture containing 2.5% glycine (Sigma-Aldrich) and 5 mM sodium chloride (Sigma-Aldrich) to achieve a concentration of 1 µg / mL. 2 mg of oxygen plasma-treated microspheres were added to 500 µL of BMP-2 solution and incubated overnight at room temperature. The microspheres were then collected by centrifugation, lyophilized, and stored at -20 °C.

[0024] Example 5: Preparation of microspheres loaded with nHA Nano-hydroxyapatite (nHA) was selected to demonstrate the ability of microspheres to load and release bioactive nanoparticles (where nHA can induce osteogenic differentiation of HMSCs). To load nHA onto the microspheres, PLL (2 mg) and nHA (50 mg) were mixed overnight in deionized water (10 mL) at 60 °C with magnetic stirring, followed by centrifugation. Microspheres I–V (10 mg) were treated with oxygen plasma to increase their surface negative charge and then immersed in the PLL-nHA mixture at 50 °C and incubated overnight with magnetic stirring at 500 rpm. The resulting nHA-loaded microspheres were washed three times with deionized water and collected after centrifugation at 2000 rpm for 5 min. The morphology of the nHA-loaded microspheres was observed by SEM. Elemental analysis was performed using an energy dispersive spectroscopy (EDS, Jeol JSM-IT200 InTouchScope, JEOL Ltd.) to evaluate the distribution of the loaded nHA on the microspheres.

[0025] Example 6: Evaluation of the formation and maintenance of PLGA microsphere-HMSC microtissues In accordance with local ethical guidelines, HMSCs were isolated from surgical waste from a single donor who had signed an informed consent form, using the method described above. HMSCs were passaged in tissue culture flasks in basal cell culture medium (α-MEM, Gibco, containing 10% v / v FBS and 10 mM ascorbic acid) until approximately 80% confluence, then digested with trypsin. To create a 3D environment for microtissue formation, U-bottom microwell arrays based on polycarbonate (PC) films were sterilized and treated to produce non-adhesive microwells. We investigated the biocompatibility of microspheres by seeding approximately 10 microspheres and 2000 HMSCs (passages 4-6) in each well and further incubating them in basal cell culture medium containing 100 U / mL penicillin and 0.1 mg / mL streptomycin (Sigma-Aldrich) at 37 °C and 5% CO2 to form microsphere-HMSC hybrid microtissues. The culture medium was replaced with fresh medium every other day. Microtissue formation was monitored over time; the formed microtissues were imaged for 14 days using an optical microscope (CKX53, Olympus), and the microtissue diameter was quantified using ImageJ analysis software. After 14 days, the metabolic activity of HMSCs was measured using the PrestoBlue cell viability assay (Thermo Fisher Scientific). Additionally, after 14 days, dead cells in the microtissues were labeled using the LIVE / DEAD fixed dead cell staining kit (Thermo Fisher Scientific) according to the manufacturer's instructions. The microtissues were then fixed with 4% w / w paraformaldehyde solution (Sigma-Aldrich) at room temperature for 30 minutes and washed three times with PBS. Next, the cells were permeabilized with Triton-X 100 (0.1% v / v) for 30 minutes and blocked with BSA (2%, w / v) for 1 hour. The microtissues were then incubated overnight at 4 °C with 33 nM phalloidin (Alexa Fluor 647, Thermo Fisher Scientific) in PBS (containing 0.1% BSA), followed by incubation at room temperature with 7 µg / mL DAPI (Sigma-Aldrich) for 45 min to label the cytoskeleton F-actin and cell nuclei, respectively. Microtissue imaging was performed using a confocal laser scanning fluorescence microscope (TCS SP8STED, Leica Microsystems) to acquire Z-axis slice stacks with a thickness of 2 µm.

[0026] Example 7: Evaluation of osteogenic activity of PLGA microspheres loaded with BMP-2 and nHA in HMSC microtissue Microspheres loaded with BMP-2 or nHA were sterilized in a UV sterilizer (256 nm, UVPTM CL-1000 UV crosslinker, Fischer Scientific) and then resuspended in osteogenic induction medium (basal medium supplemented with 10 nM dexamethasone (Sigma-Aldrich), 100 U / mL penicillin, and 0.1 mg / mL streptomycin). The microspheres were then co-inoculated with HMSCs into non-adhesive array microwells (approximately 10 microspheres / well, 2000 cells / well) and maintained in osteogenic induction medium for up to 10 days. The medium was changed every two days. Cell culture medium was collected every two days during medium changes and stored at -80 °C. Microtissue was monitored using an optical microscope. On day 10, the microtissue samples were washed with PBS buffer, fixed with paraformaldehyde (4% v / v) for 20 min, permeabilized with Triton X-100 (0.3% v / v) for 30 min, and then blocked with goat serum (5% v / v) and BSA (3% w / v) for 1 h. The microtissue samples were then incubated overnight at 4 °C with primary antibodies (anti-ALP (unconjugated rabbit recombinant polyclonal antibody, Thermo Fisher Scientific) and anti-OPN (unconjugated mouse monoclonal antibody, Thermo Fisher Scientific)). Subsequently, the samples were washed with PBS to remove residual antibodies and then incubated overnight at 4 °C with secondary antibodies (purchased from Thermo Fisher Scientific, including goat anti-rabbit IgG H&L (Alexa Fluor 488, 1 / 500) and goat anti-mouse IgG H&L (Alexa Fluor 568, 1 / 500)). Phalloidin (33 nM, Alexa Fluor 647) was added along with the secondary antibody to label the cytoskeleton F-actin. Finally, the microtissues were washed with PBS and counterstained with DAPI (7 µg / mL) to label the nuclei. The microtissues were transferred from the microwells to iBidi chamber slides and preserved in DAKO fluorescent mounting media (Agilent, USA) until imaging under a fluorescence confocal microscope by acquiring a stack of 2 µm thick Z-axis slices. ALP and OPN fluorescence intensities were quantified using ImageJ on the 3D projection confocal images at maximum intensity and normalized to the number of nuclei per microtissue.

[0027] Example 8: Assessment of endothelial budding in microsphere-HUVEC-HMSC microtissues Human umbilical vein endothelial cells (HUVECs, mixed donor, Lonza) were cultured in EGM-2 (PromoCell) medium supplemented with 100 units / mL penicillin and 100 µg / mL streptomycin. All experiments used microspheres I, II, and V, as well as HUVECs from passages 3–5. We first investigated the budding ability of HUVECs in microsphere-cell microtissues. Before seeding, HMSCs from passages 2–5 were labeled with a cell tracker (ER-Tracker Green, Invitrogen): incubated in 10 µM cell tracker solution for 1 hour, followed by washing in PBS. A mixture of approximately 40 microspheres, 800 HUVECs, and 1600 HMSCs was co-seeded into each well in EGM-2 / basal medium (1 / 1 v / v). To provide a suitable environment for HUVEC budding, the cells were incubated overnight at 37 °C and 5% CO2. The cell culture medium was then aspirated from the microwells, and 50 µL of Geltrex LDEV-Free Reduced Growth Factor basement membrane matrix (Thermo Fisher Scientific) was added to each well. A 3D gel was formed at 37 °C according to the manufacturer's instructions. After 48 hours, the microtissues were imaged using an optical microscope. The microtissues were then fixed with paraformaldehyde (4% v / v) for 20 minutes, permeabilized with Triton X-100 (0.3% v / v) for 30 minutes, and then blocked with goat serum (5% v / v) and BSA (3% w / v) for 1 hour. Samples were incubated overnight at 4 °C with CD31 primary antibody (1 µg / mL, sheep anti-human, R&D Systems, USA), and then overnight at 4 °C with donkey anti-sheep secondary antibody (1:1000, Alexa Fluor 568 nm, Invitrogen, USA). After counterstaining the cell nuclei with DAPI (7 µg / mL), microtissue imaging was performed using confocal fluorescence microscopy as described above, and the areas of CD31 and HMSCs were measured using ImageJ analysis software.

[0028] Example 9: Evaluation of HUVEC delivery in HMSC microtissues loaded with HUVECs via PLGA microspheres: Microspheres I, II, and V were co-seeded with HUVECs at different ratios (Supplementary Table S1) in EGM-2 medium into anti-adhesion wells of tissue culture plates, allowing HUVECs to be loaded onto the microspheres. After 24 hours, HUVEC-loaded microspheres prepared at a HUVEC / PLGA ratio of 10:1 were transferred to non-adhesion wells, resuspended, and co-seeded with HMSCs (1600 cells / well) labeled with a cell tracking agent as described above. The microspheres were cultured in osteogenic induction medium for up to 3 days, with the medium changed every two days. The HUVEC-loaded microspheres and the resulting co-cultured microtissues were analyzed as described above. In summary: cell nuclei, CD31, and the cytoskeleton F-actin were labeled in the HUVEC-loaded microspheres; cell nuclei and CD31 were labeled in the co-cultured microtissues. All labelings were visualized using confocal fluorescence microscopy as described above. The number of HUVECs loaded on the microspheres was determined by quantifying the number of cell nuclei on each microsphere using ImageJ analysis software. The area of ​​CD31 and HMSCs in the heterozygous microtissues was measured using ImageJ analysis software.

[0029] Statistical Analysis: Unless otherwise stated, all biological experiments were performed in n = 4 replicates. Statistical analysis was performed using Prism (GraphPad 10.3.1) software, employing unpaired Student's t-tests, one-way or two-way ANOVA, followed by Tukey HSD post-hoc tests. All data are expressed as mean ± standard deviation (SD), and significance p-values ​​are indicated as: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0030] Contents not described in detail in this specification are prior art known to those skilled in the art. Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing porous PLGA microspheres with adjustable pore size, characterized in that, Includes the following steps: (1) PLGA and porogen PVP are mixed at a weight ratio of 40 / 60 to 100 / 0 and dissolved in dichloromethane to form an oil phase, with a final polymer concentration of 200 g / L; (2) The oil phase is injected into an aqueous phase containing 0.5% PVA, with an oil-to-water volume ratio of 1% to 4%, and emulsified at 10,000–13,000 rpm for 1 minute to form an oil-in-water emulsion; (3) After evaporating dichloromethane at room temperature, freeze-dry to remove residual porogen and obtain initial porous PLGA microspheres; (4) Treat the initial microspheres with an etching solution containing 0.25 M sodium hydroxide and ethanol at a ratio of 30 / 70 v / v for 30 seconds to 5 minutes to adjust the pore size of the microspheres; (5) Filter and collect microspheres, and verify the pore size and morphology by SEM and ImageJ.

2. The method according to claim 1, characterized in that: The PLGA / PVP weight ratio is 50 / 50 to 70 / 30, and the oil-water volume ratio is 2%.

3. A porous PLGA microsphere loaded with bone repair factors, characterized in that: The surface of the microspheres described in claim 1 or 2 is treated with oxygen plasma to enhance loading efficiency, and bone morphogenetic protein BMP-2 is loaded by electrostatic adsorption, or nano-hydroxyapatite nHA is loaded by polylysine PLL. BMP-2 is a recombinant human BMP-2 protein, and nHA is nanoparticles prepared by any method.

4. The microspheres according to claim 3, characterized in that: The method for preparing the microspheres loaded with bone morphogenetic protein BMP-2 is as follows: the microspheres treated with oxygen plasma are immersed in a solution containing 1 μg / mL BMP-2, 2.5% glycine and 5 mM NaCl, and incubated overnight at room temperature.

5. The microspheres according to claim 3, characterized in that: The method for preparing the microspheres loaded with nano-hydroxyapatite (nHA) includes: Poly-L-lysine (PLL) and nHA were mixed and left to stand overnight at 60°C. The microspheres treated with oxygen plasma were immersed in a PLL-nHA mixed solution and stirred overnight at 50°C. SEM-EDS analysis showed that the Ca / P elements were evenly distributed.

6. The microspheres according to claim 3, characterized in that, The microspheres also include human umbilical vein endothelial cells (HUVECs), with each microsphere capable of carrying 5–15 HUVECs.

7. The microspheres according to claim 6, characterized in that: The microsphere loading method for the human umbilical vein endothelial cells (HUVECs) is as follows: the microspheres and HUVECs are co-cultured in a low-absorption plate at a ratio of 1:10-20 for 24 hours.

8. A method for constructing three-dimensional micro-tissues for bone regeneration, characterized in that, include: The PLGA microspheres loaded with BMP-2, nHA or HUVECs as described in claim 6 were mixed with human bone marrow mesenchymal stem cells (HMSCs) and co-seeded in a low-adsorption microarray plate. Cultured in osteogenic induction medium for 7–14 days, forming mixed self-assembled microtissues; The expression levels of ALP and OPN in the micro-tissues were significantly increased, or CD31 was formed. + Endothelial network.

9. The method according to claim 8, characterized in that, The three-dimensional microstructure is used for any of the following purposes: Construction of in vitro bone models; Cell therapy for bone defect repair; Regeneration of vascularized bone tissue; Bone-like filling material.

10. A porous microsphere system for bone regeneration, characterized in that, include: PLGA microspheres with adjustable pore size prepared by any of the methods of claims 1–2; The functionalized microspheres loaded with BMP-2, nHA, or HUVECs as described in claim 6; The three-dimensional bone microtissue formed by co-assembling the PLGA microspheres and functionalized microspheres described in any one of claims 1, 2, and 6 with HMSCs.

Images