Immunoregulation-osteogenesis bifunctional nanofiber scaffold as well as preparation method and application thereof

Abstract

The invention provides an immunoregulation-osteogenesis bifunctional nanofiber scaffold as well as a preparation method and application thereof, and belongs to the technical field of tissue engineering materials. P24 peptide is accurately encapsulated in a poly-L-lactic acid based core-shell nanofiber core by adopting a micro-sol technology, and meanwhile, an active factor SDF-1 is covalently fixed on the surface of a fiber shell. Then the outer surface of the fiber is coated with a polydopamine coating, the biological activity and adhesion of the fiber and a titanium screw are further enhanced, and finally a multifunctional screw system (S-MS-PDA-Ti) integrating mechanical and biological fixing strategies is constructed. An in-vitro research shows that the S-MS-PDA-Ti keeps good mechanical stability; the PDA coating enhances nail-holding power and relieves interface inflammation, rapid release of SDF-1 promotes collection and proliferation of MSCs, and continuous release of P24 peptide promotes formation of an osteogenesis or cartilage microenvironment. Animal experiments further prove that the material can remarkably improve the anchoring strength of screws and promote bone interface osteogenesis in a rat OP model.

Description

Technical Field

[0001] This invention belongs to the field of tissue engineering materials technology, specifically relating to an immunomodulatory-osteogenic bifunctional nanofiber scaffold, its preparation method, and its application. Background Technology

[0002] Osteoporosis (OP) is a common orthopedic disease, with a prevalence of nearly 40% in people over 50 years of age. It is projected that by 2050, the global population affected by OP will reach 60 million. Osteoporotic fractures (OPF) are the most common injury in post-traumatic osteoporotic fracture patients, with approximately 9 million new cases worldwide each year, posing a significant threat to health and imposing an economic burden. Currently, internal fixation using implants such as screws remains the preferred treatment strategy for OPF. However, OP leads to bone loss, and impaired osteogenic differentiation and directed migration of mesenchymal stem cells (MSCs) further impair screw holding power (HP). This results in implant / bone integration instability and complications such as screw loosening, severely impacting surgical outcomes. Therefore, developing implants that enhance integration at the implant / bone interface has become a major research focus.

[0003] To address this challenge, various strategies have been explored, including modifying implant materials, optimizing thread design, and modifying implant surfaces with bioactive coatings to improve fixation strength and promote osseointegration. For example, constructing titanium dioxide nanotube arrays loaded with cerium oxide nanozymes on titanium implants has been shown to alleviate oxidative stress-induced osteoblast damage and improve osseointegration in animal models of osteoporosis. However, while these methods enhance mechanical fixation, they may not completely resolve the localized bone formation defects typical of osteoporotic fractures (OPF). Insufficient recruitment of mesenchymal stem cells at the defect site can lead to inadequate new bone formation, ultimately resulting in loosening of sterile implants and delayed or nonunion of fractures.

[0004] Given the instability of the implant / bone interface and impaired healing capacity in patients with osteoporosis (OPF), it is increasingly recognized that mechanical fixation of the implant alone is insufficient in osteoporotic conditions. A key issue to address is the mechanical failure of the implant within the bone tissue, characterized by low bone volume and poor quality. Traditional fixation models, resembling a single load-bearing column, struggle to achieve effective support and stable anchorage in the fragile bone structure caused by OPF.

[0005] Researchers have already introduced polydopamine (PDA) coatings onto implant surfaces. The catechol groups in PDA exhibit strong adhesion to metal surfaces, and its inherent reactive oxygen species (ROS) scavenging and anti-inflammatory properties can significantly modulate the post-implantation immune-inflammatory microenvironment, thereby maintaining interfacial stability between the screw and bone tissue. However, while PDA improves the "mechanical fixation" of the implant, enhancing the "biological fixation" of the screw / bone interface remains crucial for treating osteoporosis (OPF).

[0006] In patients with osteoporosis, bone healing is often impaired due to local microenvironment disturbances and endogenous cell dysfunction, leading to a significant reduction in osteogenic capacity. Therefore, enhancing local MSC recruitment and osteogenic induction efficiency has become another key challenge for interfacial integration.

[0007] The dual impairment of MSC migration and osteogenic function in the OPF microenvironment is a key factor leading to osteointegration failure. As core effector cells in bone regeneration, the directed migration and functional activation of MSCs towards bone defects are crucial for initiating the bone defect repair process. During physiological bone healing, chemokines released at the injury site, such as SDF-1α and BMP-2, effectively recruit MSCs to the defect area, inducing their differentiation into osteoblasts in the osteogenic microenvironment, thus completing new bone deposition. However, under pathological osteoporosis (OP), this process is interrupted in several ways: abnormally elevated local inflammatory factors (such as TNF-α and IL-6) inhibit the expression of CXCR4 receptors on MSCs, weakening their chemotactic reactivity; simultaneously, the aging microenvironment characterized by ROS accumulation and inhibition of Wnt / β-catenin signaling significantly weakens the osteogenic differentiation potential of MSCs. This dual deficiency of both quantity and function leads to insufficient MSC density within the OPF bone defect area, ultimately depleting the "repair cell pool."

[0008] Therefore, how to develop a device that can securely fix the osteogenic differentiation of OPF with screws and effectively promote osteogenic differentiation based on the characteristics of OPF has become an urgent technical problem to be solved. Summary of the Invention

[0009] This invention aims to address the aforementioned technical problems by providing an immunomodulatory-osteogenic bifunctional nanofiber scaffold, its preparation method, and its applications. The technical objective of this invention is to develop a multifunctional nanofiber capable of adhering to the surface of titanium screws to solve the challenges of biological and mechanical fixation at the screw-bone interface. It not only enables precise spatiotemporal release of MAPK, JAK-STAT, Wnt, and bone remodeling pathways but also promotes MSC recruitment and osteogenic differentiation, rapidly establishing an early osteogenic microenvironment, thus providing a promising tissue engineering strategy for internal fixation of osteoporotic fractures.

[0010] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: This invention provides a method for preparing an immunomodulatory-osteogenic bifunctional nanofiber scaffold, comprising the following steps: (1) Hyaluronic acid solution was mixed with P24 peptide, and then mixed with Span 80 and dichloromethane and stirred to obtain hyaluronic acid microsol emulsion encapsulating P24 peptide. (2) Using poly-L-lactic acid, N,N-dimethylformamide and hyaluronic acid microsol emulsion encapsulating P24 peptide as spinning solution, fiber membranes were prepared by electrospinning, and the fiber membranes were immersed in a mixed solution of NaOH and ethanol for surface modification. (3) The surface-modified fiber membrane was activated by EDC and NHS, and then coupled and recombined with SDF-1α at 4℃ to obtain a functionalized fiber scaffold. (4) The functionalized fiber scaffold was immersed in a dopamine hydrochloride Tris-HCl solution with pH=8.5 and oscillated to deposit a PDA coating. The scaffold was then removed, cleaned, and dried to obtain an immunomodulatory-osteogenic bifunctional nanofiber scaffold.

[0011] Furthermore, the concentration of the hyaluronic acid solution in step (1) is 1 wt%.

[0012] Furthermore, the weight ratio of hyaluronic acid to P24 peptide is 100:1 (w / w).

[0013] Furthermore, the weight ratio of Span 80 to dichloromethane is 1:400.

[0014] Furthermore, in step (2), the weight ratio of poly-L-lactic acid, N,N-dimethylformamide and hyaluronic acid microsol emulsion encapsulating P24 peptide is 16:3:1 (w / w).

[0015] Furthermore, the electrospinning process parameters in step (2) are: solution feed rate 60 μl / min, applied voltage 15 ~ 20 kV, and collection distance 15 cm.

[0016] Furthermore, in step (2), the volume ratio of NaOH to ethanol is 1:1, and the concentration of NaOH is 0.25 M.

[0017] Furthermore, the surface modification process takes 2-3 minutes.

[0018] Furthermore, in step (3), the concentration of EDC is 50 mM and the concentration of NHS is 30 mM.

[0019] Furthermore, the activation treatment conditions were 25°C for 30 min.

[0020] Furthermore, the coupling and recombination reaction in step (3) takes 12 hours.

[0021] Furthermore, the concentration of dopamine hydrochloride was 2 mg / mL.

[0022] Furthermore, the pH of the Tris-HCl buffer is 8.5.

[0023] Furthermore, the condition for step (4) oscillatory deposition is to deposit at 60 rpm for 40 minutes.

[0024] A second objective of this invention is to provide an immunomodulatory-osteogenic bifunctional nanofiber scaffold prepared by the method described above.

[0025] A third objective of this invention is to provide the application of the immunomodulatory-osteogenic bifunctional nanofiber scaffold described above as a fixation material for screw implants.

[0026] This invention develops a polydopamine-functionalized coaxial electrospun nanofiber scaffold (S-MS-PDA) to address the challenges of biomechanical fixation at the screw-bone interface. The scaffold's coaxial design allows for the precise modulation of MAPK, JAK-STAT, Wnt, and bone remodeling pathways through the spatiotemporal sequential release of bioactive factors, including SDF-1 and P24. This method promotes MSC recruitment and osteogenic differentiation, rapidly establishing an early osteogenic microenvironment. Furthermore, polydopamine-mediated interface engineering creates a biomimetic transition zone, improving mechanical compatibility between the screw and bone and modulating local inflammation. In a rat osteoporosis model, the S-MS-PDA scaffold significantly reduced interfacial stress concentration and enhanced bone-implant contact by 3.31 times. This study presents a novel scaffold system combining bioactivation and mechanical reinforcement, offering a promising tissue engineering strategy for internal fixation of osteoporotic fractures.

[0027] To address the limitations of traditional osteogenic factors such as BMP-2, including potential carcinogenicity and complex synthesis, the inventors employed a low-toxicity oligopeptide, P24, derived from the functional core sequence of BMP-2. The P24 peptide retains osteogenic induction capacity while minimizing adverse effects. Furthermore, the introduction of the chemokine stromal cell-derived factor-1 (SDF-1) stimulates the migration of endogenous MSCs to the implant / bone interface, ensuring rapid early cell recruitment. By precisely designing the spatial distribution of different bioactive components within the scaffold and controlling their release kinetics, bioactive molecules can be delivered to specific targets, achieving sustained and targeted functional release.

[0028] Core-shell nanofiber structures were constructed using coaxial electrospinning technology, which effectively improved fiber stability and protected the active ingredients. P24 peptide was encapsulated within the fiber core for delayed release, while SDF-1 was conjugated on the fiber shell surface for rapid release, thus establishing a temporally and spatially coordinated release system. This sequential release strategy aims to rapidly recruit endogenous MSCs in the early stages, subsequently inducing their differentiation into osteogenic lineages to synergistically remodel the osteogenic microenvironment at the defect site, thereby achieving "biological fixation."

[0029] The beneficial effects of this invention are as follows: This invention employs microsol technology (MS) to precisely encapsulate the P24 peptide within a poly-L-lactic acid (PLLA)-based core-shell nanofiber core, while simultaneously covalently immobilizing the active factor SDF-1 on the fiber shell surface. Subsequently, a polydopamine (PDA) coating is applied to the outer surface of the fiber to further enhance its bioactivity and adhesion to titanium screws, ultimately constructing a multifunctional screw system (S-MS-PDA-Ti) integrating mechanical and biological fixation strategies. In vitro studies show that S-MS-PDA-Ti maintains good mechanical stability; the PDA coating enhances screw holding force and reduces interfacial inflammation, while the rapid release of SDF-1 promotes the recruitment and proliferation of MSCs, and the sustained release of the P24 peptide promotes the formation of osteogenic or cartilage microenvironments. Animal experiments further confirm that this material significantly improves screw anchorage strength and promotes osteogenic osteosis at the bone interface in a rat osteoporosis model. In summary, this invention provides a novel material strategy for internal fixation treatment of osteoporotic fractures by constructing a multifunctional interface system that integrates "mechanical reinforcement" and "bioactivation," and provides a theoretical and practical basis for achieving rapid integration of implant / bone interfaces and bone tissue regeneration. Figure 1 ). Attached Figure Description

[0030] Figure 1 The construction and mechanism of action of S-MS-PDA-Ti screw material; (A) the preparation strategy of S-MS-PDA-Ti; (B) based on the bridge design principle, the fixation rate of screws is improved by bio-adhesion and physical adhesion; (C) S-MS-PDA-Ti can effectively repair bone by scavenging reactive oxygen species in the microenvironment, recruiting mesenchymal stem cells and promoting their osteogenic differentiation.

[0031] Figure 2 Preparation and characterization of S-MS-PDA; (A-B) SEM and TEM images comparing conventional PLLA fibers and MS fibers, showing a distinct core-shell structure; (CD) Measurement of water contact angles showing differences in hydrophilicity between different groups; (E) Schematic diagram of pull-out test; (F) HPLC detection of release time curves of SDF and P24; (G) Stress-strain measurement and testing showing tensile strength.

[0032] Figure 3 The bioactivity of the S-MS-PDA composite scaffold was assessed. (A, B) Live / dead cell staining and quantitative analysis showed that the composite scaffold was biocompatible, scale bar = 200 μm. (C) CCK-8 assay was used to evaluate the effect of the scaffold on the proliferation of bone marrow mesenchymal stem cells. (D, E) Transwell assay was used to evaluate the ability of the scaffold to recruit bone marrow mesenchymal stem cells, scale bar = 50 μm. (F, G) Scratch assay was used to evaluate the effect of the scaffold on the migration of bone marrow mesenchymal stem cells, scale bar = 50 μm. (H) Flow cytometry analysis showed that the scaffold promoted the polarization of macrophages from M0 type to M2 type. (I) Flow cytometry analysis showed that the scaffold promoted the polarization of M1 type macrophages to M2 type.

[0033] Figure 4 S-MS-PDA promotes the expression of osteogenic-related genes and proteins; (A, B) Alkaline phosphatase (ALP) staining and quantitative analysis of ALP staining, scale bar = 200 μm; (C, D) Alizarin red staining and quantitative analysis of Alizarin red staining, scale bar = 200 μm; (EH) Immunofluorescence staining and quantification of osteogenic markers RUNX2 and OPN, scale bar = 50 μm; (I, J) Western blot analysis of OPN and RUNX2 protein expression levels.

[0034] Figure 5 For the validation of the osteoporosis model and analysis of bone microstructure; (A) Animal osteoporosis model construction scheme; (B) Representative Micro-CT three-dimensional reconstructed images for comparison of bone morphology; (C, D) Histological staining: H&E staining and Masson trichrome staining; (E) Reconstructed images of titanium screws removed from the femoral condyle of osteoporotic rats; (FH) Quantitative analysis of bone microstructure, including bone volume fraction (BV / TV), number of trabeculae (Tb.N), and trabecular connection density (Conn.D); (I) Mechanical testing: screw pull-out force and the correlation between bone-screw interface shear strength and BV / TV.

[0035] Figure 6 In vivo evaluation of multifunctional nanofibers on titanium screws and their osseointegration; (AF) Representative images and quantitative results of hard tissue sections stained with toluidine blue, HE, and Masson's trichrome, scale bar at 250 μm; (GI) Immunohistochemical detection results of type I collagen (Col-I) and osteopontin (OPN) expression.

[0036] Figure 7Transcriptome sequencing analysis; (A) Principal component analysis of differences between different groups; (B) Heatmap of the top 30 upregulated and downregulated genes; (C) Volcano plot of differentially expressed genes; (D) Winnie plot showing differential expression of key osteogenic genes; (E) GO density analysis of differentially expressed genes; (F) KEGG pathway enrichment analysis; (G-L) GSEA analysis of enriched signaling pathways between the treatment group and the control group. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention.

[0038] Example 1

[0039] I. Experimental Materials and Methods 1. Material Synthesis First, 10 mg of sodium hyaluronate powder was dissolved in 990 mg of deionized water to prepare a 1 wt% hyaluronic acid (HA) aqueous solution. This solution was then thoroughly mixed with pre-prepared P24 peptide to form a 1% HA-P24 aqueous solution. Next, 0.01 g of Span-80 was added to 4 g of dichloromethane (DCM) and stirred until homogeneous. Then, 100 μl of the HA-P24 aqueous solution was slowly added, and the mixture was stirred at high speed for 30 minutes to obtain a stable water-in-oil (W / O) emulsion system containing HA microsol particles encapsulating P24.

[0040] Subsequently, poly-L-lactic acid (PLLA) and N,N-dimethylformamide (DMF) were sequentially added to the above emulsion and stirred to prepare a microsol (MS) spinning solution. MS membranes were prepared by electrospinning (parameters: solution feed rate 60 μl / min, applied voltage 15–20 kV, collection distance 15 cm). The obtained MS membranes were immersed in a surface modification solution (25 ml of 0.25 M NaOH mixed with 25 ml of ethanol) for 2–3 minutes to cleave ester bonds and expose carboxyl groups, and then washed three times with deionized water.

[0041] Next, the fiber surface was activated for 30 min at 25 °C in MES buffer (pH=6.0) with 50 mM EDC and 30 mM NHS, and then conjugated with recombinant SDF-1α (10 μg / mL PBS) at 4 °C for 12 h. After blocking with 1 M ethanolamine (pH=8.5) for 1 h, the fiber was washed three times with PBS containing 0.05% Tween-20.

[0042] Subsequently, the functionalized fibers were immersed in Tris-HCl buffer (pH=8.5) containing 2 mg / mL dopamine hydrochloride and deposited by agitation at 60 rpm. When the coating thickness reached 45±5 nm (approximately 40 min at 25°C), the fibers were removed, washed three times with deionized water, and then dried. After immersion in the dopamine solution, the electrospun fiber membrane was tightly wound 360° onto the threads of a titanium screw.

[0043] 2. Material Characterization Scanning electron microscopy (SEM): The electrospun sample was freeze-dried, sputter-coated with a layer of gold, and observed using a Hitachi SU8010 field emission SEM at an accelerating voltage of 5 kV and a working distance of 10 mm.

[0044] Transmission electron microscopy (TEM): MS fibers were dispersed in ethanol, dropped onto a copper grid, negatively stained with phosphotungstic acid, and observed using a JEM-2100 transmission electron microscope at 200 kV.

[0045] Fourier transform infrared spectroscopy (FTIR): Spectra were recorded using a Nicolet iS50 spectrometer, from 4000 to 400 cm⁻¹. -1 , with 4 cm -1 The resolution was [resolution value], with an average of 32 scans. Samples were prepared using KBr microspheres.

[0046] Tensile strength test: Using an Instron 5967 universal testing machine, the tensile rate was 10 mm / min, the specimen was cut to 10×40 mm, and the test was repeated 5 times.

[0047] Water contact angle (WCA): Measured using a DataPhysics OCA20 system with 2 μL DI water droplets, averaged 5 times.

[0048] 3. Isolation and culture of bone marrow mesenchymal stem cells Primary bone marrow mesenchymal stem cells were isolated from 8-week-old rats using the following steps: Rats were euthanized by overdose of isoflurane. Under aseptic conditions, tibia and femur bones were harvested, and bone marrow cells were flushed out using a syringe. The collected cells were cultured in α-MEM medium supplemented with 10% fetal bovine serum (FBS) and 0.01% penicillin / streptomycin at 37°C in a 5% CO2 humidified incubator. The medium was changed every three days, and cells were passaged until 80% confluence was achieved.

[0049] 4. Osteogenic Differentiation Experiment Scaffold extracts were prepared by incubating the spun membrane in serum-free α-MEM at 37°C for 24 hours. Bone marrow mesenchymal stem cells were seeded into 24-well plates and cultured in osteogenic induction medium containing 10% fetal bovine serum, 0.01% penicillin / mycin, 50 mg / L ascorbic acid (Sigma-Aldrich, USA), 10 nM dexamethasone (Sigma-Aldrich, USA), and 10 mM β-glycerophosphate (Sigma-Aldrich, USA). The medium was changed every three days during the three-week osteogenic induction period.

[0050] 5. CCK-8 and live / dead cell assays Cell proliferation was assessed using the Cell Counting Kit 8 (CCK-8; Beyotime, China) and the Calcein / Propidium Iodide Cell Viability / Cytotoxicity Kit (Beyotime, China), all performed according to the manufacturer's instructions. Bone marrow mesenchymal stem cells were cultured at 4 × 10⁶ cells per well. 3 Cells were seeded at a density of 1,000 cells per well in 96-well plates and cultured in scaffold extract medium. CCK-8 assays and live / dead cell staining were performed on days 1, 3, and 5.

[0051] For CCK-8 assay, cells were incubated with 100 μL of culture medium containing 10 μL of CCK-8 reagent at 37°C for 1.5 hours, and then absorbance was measured at 450 nm using a FlexStation 3 microplate reader (Molecular Devices, Japan). For live / dead cell staining, cells were incubated with 250 μL of calcein-AM / propidium iodide working solution for 30 minutes and observed under a fluorescence microscope.

[0052] 6. Alkaline phosphatase (ALP) staining Seven days after osteogenic induction, alkaline phosphatase (ALP) staining was performed to assess ALP activity. Cells were fixed with 4% paraformaldehyde for 20 minutes, washed with PBS, and then incubated at room temperature with 500 μL of ALP staining solution (Beyotime, China) for 12 hours. Images were taken using a digital camera (Canon, Japan) or a microscope (Olympus, Japan).

[0053] 7. Alizarin Red Staining Following 21 days of osteogenic induction, alizarin red staining was performed to assess the formation of mineralized nodules. Cells were fixed with 4% paraformaldehyde for 20 minutes and then washed with PBS for 30 minutes. Subsequently, 500 μL of 2% alizarin red solution (Beyers, China) was added, and the cells were incubated at room temperature for 5 minutes. Images of the calcium nodules were captured using a digital camera (Canon, Japan) or a microscope (Olympus, Japan). For quantitative analysis, the calcium nodules were dissolved in 10% dodecylpyridine chloride (Sangon Biotechnology, China), and absorbance was measured at 420 nm using a microplate reader (Thermo Fisher Scientific, USA).

[0054] 8. Transwell migration experiment In the migration assay, the lower part of the Transwell chamber was injected with 600 μL of scaffold extract supplemented with 10% fetal bovine serum. Third-generation bone marrow mesenchymal stem cells were cultured for 6 hours under serum-free conditions, and then at 5 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 10 cells / mL into the upper chamber. After incubation at 37°C and 5% CO2 for 24 hours, unmigrated cells on the upper side of the membrane were removed with a cotton swab and washed with PBS. Migrated cells were fixed with 4% formalin epoxide for 20 minutes, stained with 0.1% iodine crystals for 15 minutes, washed with PBS, and then counted under a light microscope.

[0055] 9. Scratch test BMSCs were 5 × 10 5 Cells were seeded at a density of 100 cells / well in 6-well plates and cultured in α-MEM medium containing 10% FBS until the cells reached 90% confluence. A straight scratch was made using a sterile pipette tip (200 μL), and after washing with PBS, the medium containing the extract was replaced. Images of the scratched area were taken at fixed positions (100x magnification) under an inverted microscope at time points of 0, 12, and 24 hours.

[0056] 10. Immunofluorescence staining On day 7, bone marrow mesenchymal stem cells seeded on different materials were fixed with 4% paraformaldehyde for 20 minutes and treated with 0.3% Triton X-100 for 15 minutes to permeate them. After blocking with 5% BSA for 1 hour, the cells were incubated overnight at 4°C and incubated with primary antibodies against rabbit anti-Runx2 (1:200) and mouse anti-OPN (1:300). After washing with PBS, the cells were treated with secondary antibodies (1:500) of Alexa Fluor 488 in combination with anti-rabbit and Alexa Fluor 594 in combination with anti-mouse, which had been incubated in the dark for 1 hour. The nuclei were counterstained with DAPI for 5 minutes. Images were captured using a confocal microscope (Runx2: excitation / emission = 488 / 519 nm; OPN: excitation / emission = 594 / 617 nm), and the fluorescence intensity was quantitatively analyzed using ImageJ software.

[0057] 11. Animal experiments All animal experiments were approved by the Animal Care and Use Committee of Anhui Medical University. Animals were provided by the Animal Experiment Center of Anhui Medical University and housed in a standard pathogen-free environment. Rats were housed on a 12-hour light / dark cycle, with free access to food and water and ample rest. This study used 86 adult female Sprague-Daveli rats (8 weeks old, approximately 250 g). Six rats were randomly selected from these 86 rats as the sham-operated group (SHAM), where only the adipose tissue around the ovary was removed. The remaining 35 rats underwent bilateral ovariectomy (OVX) under general anesthesia to establish an osteoporosis model. Eight weeks later, osteoporosis was confirmed in randomly selected samples (n = 5 per group) using micro-computed tomography (micro-CT), hematoxylin-eosin (HE) staining, and Masson staining. Then, based on the type of coating material on the titanium screws, osteoporotic rats were randomly divided into four groups (n = 9 per group): control group, polylactic acid (PLLA) group, methyl methacrylate (MS) group, S-methyl methacrylate (S-MS) group, and S-methyl methacrylate-polyvinylbenzene (S-MS-PDA) group. Prior to implantation, the screws were appropriately coated according to standard protocol. Eight weeks after the establishment of the ovariectomy model, all rats underwent screw implantation surgery. After aseptic treatment, one side of the skin was incised to expose the distal femoral condyle. The soft tissue was carefully dissected, and a channel was drilled in the femoral condyle using Kirschner wires. Subsequently, pre-coated titanium screws (6 mm in length and 1.5 mm in diameter) were inserted into the channel according to the group assignment, ensuring accurate placement and tight fixation. All surgical procedures were performed under aseptic conditions. Postoperatively, rats received intraperitoneal injections of penicillin (80,000 units) daily for 3 days, and their overall health was monitored throughout the study.

[0058] 12. Microcomputed tomography (μCT) analysis After treatment, the rat femurs were removed and fixed in 4% paraformaldehyde for 24 hours. The samples were scanned using a Skyscan 1176 (Skyscan) μCT system (voltage: 70 kV, current: 200 μA, resolution: 9 μm / pixel). A region of interest (ROI) extending 1.35 mm from 0.45 mm below the growth plate was selected for 3D reconstruction and analysis, and relevant parameters, including bone volume / total volume (BV / TV), trabecular bone number (Tb.N), and connectivity density (Conn.D), were evaluated using CTAn software.

[0059] 13. RNA Sequencing and Analysis BMSCs were cultured to 90% confluence using α-MEM medium containing 10% fetal bovine serum. Treatment groups were then replaced with α-MEM medium containing the extract, while the control group remained unchanged. After 24 hours, cells were collected and cryopreserved in liquid nitrogen. Total RNA was extracted using Trizol reagents (Invitrogen) according to the manufacturer's protocol and submitted to the company for RNA sequencing. Bioinformatics analysis was performed using R (v4.3.2). Principal component analysis (PCA) and heatmaps were used to assess sample clustering. Differential expression analysis was performed using the "edgeR" package. Gene Ontology (GO), Kyoto Encyclopedia of Genetics and Genomes (KEGG), and GSEA analyses were performed using "clusterProfiler," and plots were generated using "ggplot2."

[0060] 14. Statistical Analysis All experiments were performed in at least three biological replicates. Data are presented as mean ± standard deviation (SD). Student's t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Statistical analysis and graph generation were performed using Prism software. A p-value < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.005).

[0061] II. Experimental Results and Analysis 1. Characterization of composite stents ( Figure 2 ) S-MS-PDA scaffolds were successfully fabricated using (microsol) electrospinning technology and observed in detail. The surface morphology of the freeze-dried electrospun fibers was observed using scanning electron microscopy. Electrospun fibers of different compositions had similar diameters, all around 400 nm, and exhibited random and discontinuous orientations. Furthermore, a dense PDA coating was observed on the fiber surface. Transmission electron microscopy (TEM) images revealed significant differences between conventional PLLA fibers and MS fibers. A distinct core-shell structure was observed in the MS fibers, with the core encapsulating P24 surrounded by an outer PLLA shell, indicating the successful synthesis of core-shell MS fibers. Simultaneously, the presence of N and S elements in the EDS spectrum confirmed the successful encapsulation of P24 and SDF-1 within the PLLA. Moreover, FITC-BSA was incorporated into the core structure of the S-MS-PDA, and fluorescence microscopy showed a significant internal fluorescence signal, further confirming the existence of a core-shell structure in the fabricated S-MS-PDA fiber.

[0062] Fourier transform infrared spectroscopy (FTIR) was then performed to evaluate the chemical structure of different scaffold components. Comparison of the FTIR spectra of PLLA and S-MS revealed a change in the absorption peak (carboxyl characteristic) near 1740 cm⁻¹ in S-MS. The depletion of carboxyl groups indicates successful grafting of SDF-1 onto the activated mass spectrometer. Furthermore, the FTIR spectra of PLLA and MS were similar, possibly because the HA and P24 components were completely encapsulated within the PLLA matrix, thus masking the characteristic HA peak. The water contact angles (WCA) of PLLA and MS fibers were 105.30°±3.39° and 112.48°±2.44°, respectively, with no statistically significant difference. However, after PDA coating, the surface contact angle of S-MS-PDA significantly decreased to 52.17°±2.77°, indicating that the PDA coating significantly improved the hydrophilicity of the scaffold. The anchoring force of the screws was measured by pull-out tests. HPLC analysis showed that SDF release was rapid, reaching nearly 70% in the first 7 days and stabilizing around day 14, with a cumulative release of nearly 80%. In contrast, P24 release was slower and more stable, gradually increasing to around 70% by day 21, exhibiting a sustained and prolonged release. Stress-strain measurements were performed to evaluate their mechanical properties. The tensile strengths of the PLLA, MS, S-MS, and S-MS-PDA membranes were 4.52±0.57, 3.95±0.29, 3.87±0.22, and 4.74±0.34 MPa, respectively. Compared to the PLLA membrane, the maximum tensile strength of the MS membrane was slightly lower, possibly due to the presence of the core-shell structure. However, the mechanical properties of the S-MS-PDA membrane were significantly improved, which can be attributed to the deposition and reinforcement of the nanofiber structure.

[0063] 2. Bioactivity of composite scaffolds ( Figure 3 ) Electrospun fibers serve as the direct contact interface between titanium screws and bone, thus requiring excellent biocompatibility. Otherwise, they may lead to cell death at the implant / bone interface, triggering an immune response and ultimately impairing bone healing. To assess biocompatibility, S-MS-PDA fibers were soaked in culture medium for 24 hours to obtain a material extract, which was then co-cultured with BMSCs. Cell viability was assessed using live / dead staining and CCK-8 assay. After 24 hours of co-culture, most cells remained viable, with no statistically significant differences between groups, and survival rates exceeding 90% in all groups. Similarly, CCK-8 assays showed that BMSC proliferation continued to increase at 1, 3, and 5 days. However, cell proliferation in the MS and S-MS-PDA groups was significantly faster than in the Con group. These results indicate that the synthetic fiber coating did not inhibit the proliferation of bone marrow mesenchymal stem cells and possessed good biocompatibility. The recruitment effect of the material on bone marrow mesenchymal stem cells was evaluated using Transwell migration and scratch assays. Compared with the control group and the unmodified scaffold, the S-MS-PDA group significantly enhanced BMSC migration and scratch healing, demonstrating superior chemotaxis and regenerative potential. In contrast, PLLA and MS alone showed limited effects. Polydopamine (PDA) is known to reprogram macrophages from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype. During material implantation, foreign body stimulation and the osteoporotic (OP) microenvironment often induce macrophage polarization towards the M1 phenotype, leading to the release of reactive oxygen species (ROS) and disrupting bone healing. Therefore, promoting macrophage reprogramming from M1 to M2 is considered beneficial for improving the bone regeneration microenvironment.

[0064] To investigate this effect, we analyzed macrophage polarization using flow cytometry. After 7 days of culture, the expression of the M2 macrophage marker CD206 in the S-MS-PDA group was significantly higher than that in the Con group, indicating that this material has the ability to promote M2 polarization. Furthermore, even after LPS-induced M1 polarization, S-MS-PDA treatment could still effectively reprogram M1 macrophages to the M2 phenotype. In conclusion, S-MS-PDA can not only promote the polarization of undifferentiated (M0) macrophages to the M2 phenotype but also promote the reprogramming of pro-inflammatory M1 macrophages to M2 cells, which may help improve the inflammatory microenvironment and enhance bone tissue regeneration.

[0065] 3. Osteogenic effect of composite scaffolds ( Figure 4 ) The osteogenic effect of nanofibers on bone marrow mesenchymal stem cells (BMSCs) was evaluated using alkaline phosphatase (ALP) activity assays and Alizarin Red staining. After 7 days of culture, the ALP activity of BMSCs in the MS and S-MS-PDA groups was significantly higher than that in the PLLA group. ALP staining results showed that electrospun fibers containing P24 had a significant positive effect on ALP activity in BMSCs. Subsequent quantitative analysis of ALP activity further confirmed these results. Alizarin Red staining was performed on day 21 to further evaluate the effect of different components on osteogenic differentiation of BMSCs. In BMSCs cultured on MS and S-MS-PDA scaffolds, the density of calcium nodules was higher, indicating higher osteogenic activity. Calcium nodules were dissolved in perchloric acid, and absorbance was measured at 420 nm. Statistical results showed that the absorbance values ​​of the MS and S-MS-PDA groups were significantly higher than those of the group without P24, further confirming the osteogenic effect of P24.

[0066] Immunofluorescence staining of RUNT-associated transcription factor 2 (Runx2) and osteopontin (OPN) was performed on day 7. Semi-quantitative analysis of the fluorescence images showed that the fluorescence intensity of the MS and S-MS-PDA groups was approximately twice that of the group without P24. Runx2 protein is mainly expressed in osteoblasts and is a key regulator of bone development and maintenance, while OPN expression also indicates bone formation. Therefore, the fluorescence results indicated that nanofibers containing P24 significantly promoted bone formation. Western blotting further confirmed that the expression levels of OPN and RUNX2 proteins in the MS and S-MS-PDA groups were significantly higher than those in the other two groups.

[0067] 4. Establishment of an in vivo osteoporosis model ( Figure 5 ) Following the pre-established protocol, an osteoporosis model was established in SD rats through ovariectomy (OVX). Eight weeks post-surgery, micro-CT scans were performed to confirm the occurrence of osteoporosis in the sham-operated group and the OVX group. Micro-CT analysis showed that, compared to the sham-operated group, OVX rats exhibited significantly reduced bone volume, deteriorated trabecular structure, and enlarged medullary cavity in the femoral condyle. Furthermore, histological evaluation using H&E and Masson trichrome staining confirmed significant morphological differences between the groups. Specifically, the sham-operated group showed normal lamellar trabecular structure, while OVX rats showed degeneration of rod-shaped trabecular bone. Quantitative analysis of CT data further revealed significant differences between the two groups in bone mineral density (BMD), bone volume (BV), connectivity density (Conn.D), trabecular number (Tb.N), and trabecular thickness (Tb.Sp), confirming the successful establishment of the osteoporosis model and demonstrating statistically significant differences compared to the sham-operated group.

[0068] 5. In vivo osteogenic effect of composite scaffolds ( Figure 5 ) Eight weeks after ovariectomy, titanium screws were implanted into the femoral condyles of all osteoporotic rats. In this study, the group with direct screw implantation was designated as the control group. Before screw implantation, PLLA, MS, S-MS, and S-MS-PDA materials were coated onto the screw surface and then implanted together into the femoral condyle screw channel, corresponding to the PLLA, MS, S-MS, and S-MS-PDA groups, respectively. The screws were accurately fixed to the femoral condyle, and the size of the titanium screws precisely matched the rat's femoral condyle. Eight weeks after implantation, all rats were sacrificed, and the femoral condyles were harvested for Micro-CT analysis to assess peri-screw bone formation. Eight weeks later, the BV / TV percentage in the S-MS-PDA group was significantly higher than in the other groups. This result may be related to the stable adhesion of S-MS-PDA within the screw channel, its regulation of local macrophage polarization, and its effective recruitment and differentiation of bone marrow mesenchymal stem cells, thereby promoting bone integration. The Micro-CT results were also satisfactory, showing that the BV / TV value (9.063±0.531%) was highest in the S-MS-PDA group, with the highest Tb.N value (3.247±0.249 1 / mm) and Conn.D value (25.440±1.634 1 / mm3), compared to 3.16, 1.74, and 2.306 times that of the control group, respectively. In addition, we evaluated the screw anchorage strength through a tooth extraction test. The results showed that 8 weeks after implantation, the extraction force of the screw in the S-MS-PDA group was the highest (89.074±4.441 N), which was 1.238, 1.787, 2.064, and 2.171 times that of the S-MS group (71.928±6.118 N), MS group (49.844±4.367 N), PLLA group (43.14±3.167 N), and control group (41.03±1.927 N), respectively. Figure 5 ).

[0069] Subsequently, osteogenicity was further assessed by histopathological examination (H&E, Masson staining, toluidine blue, and immunohistochemistry). By week eight, compared with the control group, the S-MS-PDA group showed significantly enhanced new bone formation and richer trabecular structure around the screws. Quantitative analysis showed that the H&E staining area in the S-MS-PDA group (55.358±3.422%) was significantly increased, 3.259 times that of the control group (16.986±0.448). A similar trend was observed in Masson staining. In addition, the bone-implant contact (BIC) in the S-MS-PDA group was 3.311 times that of the control group. Finally, the protein expression levels of type I collagen and osteopontin (OPN) in each group were assessed by immunohistochemistry. The results showed that the expression levels of both osteogenic marker proteins were significantly increased in the S-MS-PDA group compared with other groups. Figure 6 ).

[0070] In summary, pathological examination of animal tissue sections successfully demonstrated that S-MS-PDA exhibits optimal osteogenic performance. This indicates that the strategy of macrophage reprogramming to recruit and differentiate bone marrow mesenchymal stem cells can be effective in vivo.

[0071] 6. Transcriptomic analysis of bone marrow mesenchymal stem cells (BMSCs) Figure 7 ) PCA analysis and heatmap results showed significant differences between the two groups, indicating that the extract effectively altered the biological function of bone marrow mesenchymal stem cells (BMSCs). Simultaneously, volcano plots and box plots revealed significant upregulation of three key osteogenic-related genes in the treatment group, confirming that the material effectively promoted osteogenic differentiation of BMSCs. Furthermore, GO and KEGG enrichment analyses showed that differentially expressed genes were mainly enriched in pathways such as Wnt, MAPK, and bone growth, suggesting that the material may promote BMSC osteogenic differentiation through these pathways. Finally, GSEA analysis further confirmed that the material can activate signaling pathways including MAPK, JAK-STAT, Wnt, and bone remodeling. Therefore, bioinformatics analysis results indicate that this biomaterial can promote BMSC osteogenic differentiation through multiple signaling pathways.

[0072] III. Conclusion This invention, based on the "structure-function" synergistic design concept, successfully constructs a multifunctional composite material (S-MS-PDA-Ti) that promotes implant / bone interface integration. Specifically, this invention embeds the osteogenic active peptide P24 within a poly-L-lactic acid (PLLA) core, forming a core-shell structured microsol-electrospun nanofiber (MS). Matrix cell-derived factor-1α (SDF-1α) is chemically grafted onto the fiber surface, and a polydopamine (PDA) coating is applied to stabilize the bond with titanium screws. This design employs a three-layer functionalization strategy: rapid surface release of SDF-1α to recruit MSCs, sustained core release of P24 peptide to induce osteogenic differentiation, and PDA-mediated interface regulation of the immune microenvironment—precisely targeting the core mechanism of osteoporotic fracture (OPF) bone integration failure.

[0073] In this invention, the innovative spatiotemporal controlled-release design of S-MS-PDA-Ti achieves precise recruitment and directed differentiation of endogenous MSCs: early and rapid release of SDF-1α mimics physiological damage signals, activating the CXCR4 / SDF-1α axis and efficiently recruiting MSCs from the bone marrow and peripheral circulation to the bone-screw interface; continuous release of P24 peptide continuously activates the BMP / Smad pathway, effectively promoting osteogenic differentiation of MSCs. Importantly, the introduction of the PDA coating significantly inhibits the activation of pro-inflammatory M1 macrophages by clearing ROS and regulating the NF-κB signaling pathway, while promoting M2 macrophage polarization, thereby reversing the local microenvironment from a pro-inflammatory state to a repair-dominated state, creating an "immuno-osteogenic dual-regulatory" stem cell niche. Compared with single-factor delivery systems, this strategy greatly accelerates bone regeneration at the OPF screw fixation site, further confirming the synergistic necessity of mesenchymal stem cell functional recruitment and immune microenvironment remodeling for OPF bone regeneration.

[0074] In summary, this invention successfully prepared a multifunctional nanofiber capable of adhering to the surface of a titanium screw based on electrospinning technology. This nanofiber can reprogram macrophages towards M2 polarization and recruit and differentiate bone marrow mesenchymal stem cells in situ within the bone marrow cavity, providing strong support for bone repair in OPF. Therefore, the S-MS-PDA-Ti prepared in this invention has the potential to treat OPF.

[0075] Comparative Example 1 The composite fiber scaffold was prepared according to the method of Example 1, except that the hyaluronic acid solution was replaced with a gelatin solution. The results showed that due to gelatin's stronger hydrophilicity, poor film-forming properties, and insufficient stability in the oil phase, the resulting microsol emulsion structure was unstable. The droplets easily fused and broke during electrospinning, leading to a significantly rough fiber morphology, uneven diameter distribution, and difficulty in forming a stable core-shell structure. Simultaneously, the encapsulation efficiency of P24 peptide in the gelatin system was significantly reduced, with a noticeable burst release phenomenon in the early stages, failing to achieve the expected sustained-release effect. Furthermore, the insufficient exposure of carboxyl groups on the surface of this comparative scaffold reduced the subsequent EDC / NHS coupling efficiency and decreased the grafting amount of SDF-1α, resulting in significantly weaker BMSC migration ability and osteogenic induction effect compared to the hyaluronic acid system of this invention. Therefore, using gelatin instead of hyaluronic acid significantly weakens the fiber structure stability and bioactivity, which is detrimental to achieving the dual functions of immunomodulation and osteogenic synthesis of this invention.

[0076] Comparative Example 2 The composite fiber scaffold was prepared according to the method in Example 1, except that the P24 peptide was replaced with an equimolar amount of other commonly used bioactive peptides (RGD peptide). The results showed that, because this peptide lacks the BMP-2 functional core sequence unique to P24, its activity in promoting osteogenic differentiation of BMSCs was significantly insufficient. Even when successfully encapsulated in a microsol emulsion, it failed to effectively upregulate osteogenic-related proteins such as Runx2 and OPN. Furthermore, its ability to induce ALP activity and promote calcium nodule formation was significantly weaker than the P24 system of this invention. More importantly, these substitute peptides easily interacted non-specifically with hyaluronic acid during electrospinning, resulting in reduced microsol structural stability, local collapse or voids in the fiber morphology, further affecting the sustained drug release performance and causing significant early burst release. Ultimately, this comparative material was significantly inferior to the present invention using the P24 peptide in terms of in vitro cell migration, osteogenic induction, and interfacial binding strength, failing to achieve the overall technical effect of synergistic immune regulation and osteogenic dual functions.

Claims

1. A method for preparing an immunomodulatory-osteogenic bifunctional nanofiber scaffold, characterized in that, Includes the following steps: (1) Hyaluronic acid solution was mixed with P24 peptide, and then mixed with Span 80 and dichloromethane and stirred to obtain hyaluronic acid microsol emulsion encapsulating P24 peptide. (2) Using poly-L-lactic acid, N,N-dimethylformamide and hyaluronic acid microsol emulsion encapsulating P24 peptide as spinning solution, fiber membranes were prepared by electrospinning, and the fiber membranes were immersed in a mixed solution of NaOH and ethanol for surface modification. (3) The surface-modified fiber membrane was activated by EDC and NHS, and then coupled and recombined with SDF-1α at 4℃ to obtain a functionalized fiber scaffold. (4) The functionalized fiber scaffold was immersed in a dopamine hydrochloride Tris-HCl solution with pH=8.5 and oscillated to deposit a PDA coating. The scaffold was then removed, cleaned, and dried to obtain an immunomodulatory-osteogenic bifunctional nanofiber scaffold.

2. The preparation method according to claim 1, characterized in that, The concentration of the hyaluronic acid solution in step (1) is 1 wt%; preferably, the weight ratio of hyaluronic acid to P24 peptide is 100:1; more preferably, the weight ratio of Span 80 to dichloromethane is 1:

400.

3. The preparation method according to claim 1, characterized in that, In step (2), the weight ratio of poly-L-lactic acid, N,N-dimethylformamide and hyaluronic acid microsol emulsion encapsulating P24 peptide is 16:3:

1.

4. The preparation method according to claim 1, characterized in that, The process parameters for electrospinning in step (2) are: solution feed rate 60 μl / min, applied voltage 15 ~ 20 kV, and collection distance 15 cm.

5. The preparation method according to claim 1, characterized in that, In step (2), the volume ratio of NaOH to ethanol is 1:1, and the concentration of NaOH is 0.25 M; preferably, the surface modification treatment time is 2-3 minutes.

6. The preparation method according to claim 1, characterized in that, In step (3), the concentration of EDC is 50 mM and the concentration of NHS is 30 mM; preferably, the activation treatment is performed at 25°C for 30 min.

7. The preparation method according to claim 1, characterized in that, The coupling and recombination reaction in step (3) takes 12 hours; preferably, the concentration of dopamine hydrochloride is 2 mg / mL; more preferably, the pH of the Tris-HCl buffer is 8.

5.

8. The preparation method according to claim 1, characterized in that, Step (4) The condition for oscillatory deposition is to deposit at 60 rpm for 40 minutes.

9. An immunomodulatory-osteogenic bifunctional nanofiber scaffold prepared by the method according to any one of claims 1-8.

10. The use of the immunomodulatory-osteogenic bifunctional nanofiber scaffold of claim 9 as a fixation material for screw implants.

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