Composite scaffold for promoting regeneration of diabetic bone defects and preparation method and application thereof

Abstract

This invention discloses a composite scaffold for promoting bone regeneration in diabetic patients, its preparation method, and its applications, relating to the field of medical biomaterials technology. The composite scaffold for promoting bone regeneration in diabetic patients comprises: a three-dimensional porous framework; and a bioactive hydrogel layer loaded with naringin. The bioactive hydrogel layer loaded with naringin is coated on the surface and pore walls of the three-dimensional porous framework. The technical solution provided by this invention uses a 3D-printed PLGA / HA porous framework to provide mechanical support and osteoconductivity, and a supramolecular hydrogel coating loaded with naringin to endow it with immunomodulatory and drug sustained-release functions. This scaffold has good hydrophilicity and biocompatibility, and can effectively induce macrophage polarization towards the M2 anti-inflammatory phenotype by activating the JAK-STAT signaling pathway, improving the local inflammatory microenvironment of diabetic bone defects. Simultaneously, it indirectly promotes vascular endothelial cell migration, lumen formation, and osteogenic differentiation of bone marrow mesenchymal stem cells by regulating macrophages.

Description

Technical Field

[0001] This invention relates to the field of medical biomaterials technology, and in particular to a composite scaffold for promoting bone regeneration in diabetic patients, its preparation method, and its application. Background Technology

[0002] Bone tissue engineering offers new approaches to treating bone defects caused by trauma, infection, or tumor resection; among them, 3D printing technology, which can precisely construct personalized bone repair scaffolds with biomimetic structures according to the patient's defect morphology, has become a research hotspot in this field.

[0003] However, for diabetic patients, bone repair scaffolds often fail to achieve ideal bone repair results. The diabetic microenvironment is characterized by persistent hyperglycemia and chronic inflammation, which triggers a series of pathological reactions: on the one hand, hyperglycemia inhibits vascular endothelial cell function, leading to reduced angiogenesis and insufficient blood supply to the bone defect area; on the other hand, the persistent inflammatory response causes macrophages to polarize towards the pro-inflammatory M1 phenotype, making it difficult for them to transform into the anti-inflammatory and pro-repair M2 phenotype. Inflammatory factors secreted by M1 macrophages not only inhibit osteogenic differentiation of bone marrow mesenchymal stem cells but also further disrupt the balance between bone formation and bone resorption, severely hindering the healing process of bone defects. Therefore, how to regulate the local microenvironment of diabetic bone defects, guide macrophages to polarize from the M1 to the M2 phenotype, and promote angiogenesis is an urgent problem to be solved. Summary of the Invention

[0004] The main objective of this invention is to propose a composite scaffold for promoting bone regeneration in diabetic patients, its preparation method, and its application, aiming to promote the repair of bone defects in diabetic patients.

[0005] To achieve the above objectives, the present invention proposes a composite scaffold for promoting bone regeneration in diabetic patients, comprising: Three-dimensional porous framework; Bioactive hydrogel layer loaded with naringin; The bioactive hydrogel layer loaded with naringin is coated on the surface and inner walls of the pores of the three-dimensional porous framework.

[0006] In one embodiment, the three-dimensional porous framework is composed of polylactic acid-glycolic acid copolymer and hydroxyapatite.

[0007] In one embodiment, the mass ratio of hydroxyapatite to polylactic acid-glycolic acid copolymer is 1:4.

[0008] In one embodiment, the bioactive hydrogel layer is a supramolecular hydrogel formed by crosslinking gelatin and acrylic β-cyclodextrin under ultraviolet light.

[0009] In one embodiment, the concentration of gelatin in the supramolecular hydrogel is 8% (w / v), and the concentration of acrylic β-cyclodextrin is 10% (w / v).

[0010] In one embodiment, the loading concentration of naringin in the bioactive hydrogel layer is 100 μM.

[0011] In one embodiment, the three-dimensional porous skeleton is fabricated by 3D printing.

[0012] This invention also proposes a method for preparing a composite scaffold that promotes bone regeneration in diabetic patients, comprising the following steps: S1. Prepare a three-dimensional porous framework; S2. Preparation of bioactive hydrogel loaded with naringin; S3. The bioactive hydrogel loaded with naringin is coated on the surface of the three-dimensional porous framework and cross-linked by ultraviolet light irradiation to obtain the composite scaffold.

[0013] In one embodiment, step S1 is specifically as follows: Hydroxyapatite and polylactic acid-glycolic acid copolymer were mixed at a mass ratio of 1:4 and dissolved in 1,4-dioxane to prepare a 10% (w / v) homogeneous slurry. The slurry was then 3D printed and freeze-dried to obtain the three-dimensional porous skeleton.

[0014] This invention also proposes the application of composite scaffolds that promote the regeneration of diabetic bone defects in the preparation of drugs or medical devices for the treatment of diabetic bone defects.

[0015] The composite scaffold of this invention provides mechanical support and osteoconductivity using a 3D-printed PLGA / HA porous framework, while a supramolecular hydrogel coating loaded with naringin endows it with immunomodulatory and drug-release functions. This scaffold exhibits good hydrophilicity and biocompatibility, effectively inducing macrophage polarization towards the M2 anti-inflammatory phenotype by activating the JAK-STAT signaling pathway, thus improving the inflammatory microenvironment at the site of diabetic bone defects. Simultaneously, it indirectly promotes vascular endothelial cell migration, lumen formation, and osteogenic differentiation of bone marrow mesenchymal stem cells by regulating macrophages. In vitro and in vivo experiments have demonstrated that this scaffold achieves multiple synergistic functions of immunomodulation, angiogenesis, and osteogenic development, significantly accelerating bone defect repair in diabetic patients and providing an effective therapeutic strategy for diabetic bone regeneration. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. The data in the drawings are expressed as mean ± standard deviation. After one-way ANOVA, Dunnett test was performed, and *P<0.05, **P<0.01, ***P<0.001. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0017] Figure 1 The diagram shows the PHSN scaffold and the mechanism of bone formation regulation. (A) is the flowchart of PH scaffold preparation, (B) is the flowchart of PHSN scaffold modification, and (C) is the bone repair effect of PHSN scaffold in vivo. Figure 2 The characterization of different scaffolds is shown; (A) is a morphological observation image, (B) is an electron microscope (SEM) observation image, (C) is an energy dispersive spectroscopy (EDS) image, (D) is a water contact angle measurement result image, (E) is an ion mobility spectrometry (FT-IR) analysis image, (F) is a degradation rate graph, (G) is a graph of cumulative limonene release, (H) is a graph of calcium ion release, (I) is a graph of phosphorus release, and (J) is a statistical analysis image of water contact angle. Figure 3 The cytotoxic effects of naringin and the biocompatibility of different scaffolds are shown. Among them, (A) is the result of calcein / propidium iodide staining, (B) is the result of CCK-8 experiment, (C) is the cell survival rate statistics, (D) is the scaffold stained with calcein / propidium iodide, (E) is the cell survival rate statistics on the scaffold, and (F) is the result of phalloidin staining. Figure 4 The in vitro response of macrophages to the scaffold is shown in the following diagrams: (A) Immunofluorescence staining of M2 marker (CD206), (B) Immunofluorescence staining of M1 marker (iNOS), (C) Statistical analysis of CD206 staining results, (D) Statistical analysis of iNOS staining results, (E) mRNA expression levels of iNOS, IL-6, and IL-23 genes, and (F) mRNA expression levels of CD206, Arg-1, and IL-10 genes. Figure 5The study showcases the migration, angiogenesis, and osteogenic properties of different scaffolds in vitro. (A) is a migration map of HUVECs, (B) is a statistical graph of migration rate, (CD) is a statistical graph of node number and total length, (E) is a graph of tube formation experiment results, (F) is a statistical graph of ALP-positive regions, (H) is a graph of ARS staining, (G) is a statistical graph of ARS-positive regions, (I) is a graph of ALP staining, and (J) is a graph of mRNA expression levels of ALP, cyanate, and Runx2 genes. Figure 6 The in vivo osteogenic effect of different scaffolds is shown. (A) is a schematic diagram of different scaffold implantation, (B) is a representative 3D micro-CT image at 4 and 8 weeks postoperatively, (C) is a statistical graph of regenerated bone volume, (DE) is a statistical graph of bone volume / bone surface area (BV / TV) and bone mineral density (BMD), (F) is a hematoxylin-eosin (HE) staining image, and (G) is a Masson staining image; NB in ​​the figure represents new bone. Figure 7 The study demonstrates the in vivo osteogenic and osteoclastogenic properties of different scaffolds under diabetic conditions. (A) is an ALP immunohistochemical staining image, (B) is an ALP staining statistical graph, (C) is a cyanate immunohistochemical staining image, (D) is a cyanate staining statistical graph, (E) is a TRAP staining image, and (F) is a TRAP-positive area statistical graph. In the figure, ▲ indicates a staining-positive area, and * indicates a 3D printed scaffold. Figure 8 This study demonstrates the in vivo immunomodulatory and angiogenic properties of different scaffolds under diabetic conditions. (A) shows the immunofluorescence staining patterns of M0 and M2 markers (F4 / 80 and CD206), (B) shows the statistical analysis of CD206 staining results, (C) shows the immunofluorescence staining patterns of M0 and M1 markers (F4 / 80 and iNOS), (D) shows the statistical analysis of iNOS staining results, (E) shows the immunofluorescence staining pattern of the angiogenic marker (CD31), and (F) shows the statistical analysis of CD31 staining results. ▲ indicates a positive staining area, and * indicates a 3D-printed scaffold. Figure 9The transcriptome sequencing and signaling pathway analysis results are shown in the following diagrams: (A) Pearson correlation analysis results between samples; (B) Volcano plot of differentially expressed genes; (C) Heatmap and hierarchical clustering analysis of differentially expressed genes; (DF) Heatmap analysis of differentially expressed genes related to macrophage polarization, cell migration and cell differentiation; (G) Distribution of GO-enriched upregulated terms in the control group and PHSN group; (H) Distribution of KEGG-enriched pathways in the control group and PHSN group; (I) Network analysis of differentially expressed gene protein interaction; (J) GSEA analysis of JAK-STAT signaling pathway; and (K) Western-Blot analysis of JAK-STAT signaling pathway-related proteins.

[0018] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. 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 a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] It should be noted that if the embodiments of the present invention involve directional indicators (such as up, down, left, right, front, back, etc.), the directional indicators are only used to explain the relative positional relationship and movement of the components in a specific posture. If the specific posture changes, the directional indicators will also change accordingly.

[0021] Furthermore, if the embodiments of this invention involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the use of "and / or" or "and / or" throughout the text includes three parallel solutions. For example, "A and / or B" includes solution A, solution B, or a solution where both A and B are satisfied simultaneously. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by this invention.

[0022] Diabetes disrupts the normal bone healing process, primarily due to oxidative stress and inflammation caused by high blood sugar. High blood sugar not only inhibits angiogenesis but also exacerbates local inflammation, thus interfering with bone repair. This pro-inflammatory environment inhibits the osteogenic capacity of bone marrow mesenchymal stem cells, a key reason for impaired bone regeneration in diabetic patients.

[0023] Macrophages play a central role in this process. They can differentiate into a pro-inflammatory M1 phenotype or an anti-inflammatory M2 phenotype based on environmental signals. M2 macrophages can suppress inflammation and promote tissue repair; therefore, inducing macrophage polarization towards the M2 phenotype at bone defect sites is considered a promising strategy for treating diabetic bone defects.

[0024] Specifically, hyperglycemia and inflammation in the diabetic microenvironment hinder the healing process of bone defects in diabetic patients. Due to persistent inflammation, the phenotypic transformation of M1 to M2 anti-inflammatory macrophages is slowed, and angiogenesis is also reduced.

[0025] Naringin is a natural flavanone compound with anti-inflammatory and anti-osteoporosis activities. It can effectively inhibit key inflammatory factors such as TNF-α and IL-1β, and promote the production of M2 macrophages, showing great potential in regulating immunity.

[0026] In the field of bone repair materials, 3D printing technology can precisely construct biomimetic scaffolds. Polylactic-co-glycolic acid copolymer (PLGA) is commonly used as a bone scaffold material due to its good biocompatibility and biodegradability; its bone-forming ability is further enhanced after being combined with hydroxyapatite (HA). Supgels, composed of gelatin and acrylic β-cyclodextrin, possess excellent self-repair capabilities and drug delivery performance, effectively promoting cell recruitment and differentiation. However, hydrogels have relatively weak mechanical strength and are difficult to support bone defects alone.

[0027] Reference Figure 1 This invention proposes combining a PLGA / HA scaffold with SupGels hydrogel loaded with naringin. This design retains the scaffold's supporting structure while endowing it with immunomodulatory and drug-release functions, and is expected to provide a new approach to the treatment of diabetic bone defects by improving the local microenvironment and synergistically promoting angiogenesis and osteogenic formation.

[0028] This invention proposes a composite scaffold for promoting bone regeneration in diabetic patients, comprising a three-dimensional porous framework and a bioactive hydrogel layer loaded with naringin; wherein the bioactive hydrogel layer loaded with naringin is coated on the surface and inner walls of the pores of the three-dimensional porous framework. The composite scaffold proposed in this invention retains the mechanical support capacity of the framework while endowing the scaffold with immunomodulatory and drug-loading functions through the hydrogel layer, laying the foundation for achieving a synergistic effect of microenvironment regulation and bone repair in diabetic bone defects.

[0029] Furthermore, the three-dimensional porous framework is composed of polylactic acid-glycolic acid copolymer and hydroxyapatite. The use of PLGA and HA composites to construct the three-dimensional porous framework leverages the advantages of PLGA's good biocompatibility and controllable degradation rate, while also introducing the osteoconductivity and osteogenic activity of HA, significantly enhancing the scaffold's bone repair potential. Correspondingly, the mass ratio of HA to PLGA is 1:4. This ratio ensures the scaffold's mechanical strength while providing a suitable osteoconducting microenvironment, which is conducive to the adhesion, proliferation, and osteogenic differentiation of bone marrow mesenchymal stem cells.

[0030] The three-dimensional porous skeleton is made through 3D printing. The three-dimensional porous skeleton can be prepared by 3D printing technology and can be customized according to the patient's bone defect morphology. It can construct interconnected channels with biomimetic structure, which is conducive to cell ingrowth and nutrient exchange.

[0031] In this invention, the bioactive hydrogel layer is a supramolecular hydrogel, which is formed by cross-linking gelatin and β-cyclodextrin with acrylic acid under ultraviolet light. The supramolecular hydrogel formed by cross-linking gelatin and β-cyclodextrin with acrylic acid under ultraviolet light possesses excellent biocompatibility, self-healing ability, and sustained-release properties, making it an ideal carrier for naringin and enabling its long-term, controllable release at the defect site.

[0032] Furthermore, in the supramolecular hydrogel, the concentration of gelatin is 8% (w / v), and the concentration of acrylic β-cyclodextrin is 10% (w / v). At this ratio, the formed hydrogel network structure is stable, and it maintains good coating performance and drug release behavior even after being loaded with naringin.

[0033] In addition, the loading concentration of naringin in the bioactive hydrogel layer is 100 μM. This concentration can effectively promote cell proliferation and M2 macrophage polarization without cytotoxicity, thus achieving a balance between safety and functionality.

[0034] This invention also proposes a method for preparing a composite scaffold that promotes bone regeneration in diabetic patients, comprising the following steps: S1. Prepare a three-dimensional porous framework; S2. Preparation of bioactive hydrogel loaded with naringin; S3. The bioactive hydrogel loaded with naringin is coated on the surface of the three-dimensional porous framework and cross-linked by ultraviolet light irradiation to obtain the composite scaffold.

[0035] Step S1 is as follows: Hydroxyapatite and polylactic acid-glycolic acid copolymer were mixed at a mass ratio of 1:4 and dissolved in 1,4-dioxane to prepare a 10% (w / v) homogeneous slurry. This slurry was then 3D printed and freeze-dried to obtain the three-dimensional porous framework. The scaffold obtained in this step has a uniform pore structure and stable mechanical properties, making it suitable for repairing bone defects in load-bearing areas.

[0036] The composite scaffold preparation method provided by this invention, through a step-by-step strategy of first printing the scaffold and then coating it with a drug-loaded hydrogel, avoids the inactivation of active drugs during high-temperature or organic solvent treatment, thus protecting the bioactivity of naringin.

[0037] This invention also proposes the application of the above-mentioned composite scaffold for promoting the regeneration of diabetic bone defects in the preparation of drugs or medical devices for treating diabetic bone defects. The composite scaffold of this invention can effectively improve the local inflammatory microenvironment of diabetic bone defects and accelerate the bone regeneration process through the synergistic effects of multiple functions such as immune regulation, angiogenesis and osteoproliferation. It can be used to prepare drugs or medical devices for treating diabetic bone defects and has good prospects for clinical translation.

[0038] The following detailed description is provided in conjunction with examples and verification cases. Example Fabrication of microporous PHSN scaffolds PLGA (75:25, BIOMQ, Shenzhen, China) was dissolved in 1,4-dioxane (Aladdin, China) to prepare a 10% (w / v) homogeneous solution. HA (hydroxyapatite, Mqsw210301, BIOMQ, Shenzhen, China) was then added to the PLGA solution, resulting in a final mass ratio of 1:4 (HA:PLGA, w / w). The PLGA / HA mixture was homogenized using a magnetic stirrer until a uniform liquid paste was obtained. Composite scaffolds were fabricated using a 3D printer (Bio-Architect@WS, regenovo, Hangzhou, China) with a 27G nozzle, and the printing speed, pressure, and platform temperature were set to 45 mm / s, 0.1 MPa, and -20°C, respectively. All scaffolds were then freeze-dried for 24 hours in a freeze dryer (BoYiKangFD-1-50, Beijing, China) at a vacuum pressure of 20–40 Pa. The final scaffold composed of PLGA and HA was named PH.

[0039] 10 g of β-cyclodextrin (β-CD, catalog number #HY-107201, MCE Pharmaceuticals, USA) was dissolved in 150 mL of dimethylformamide (DMF, Fisher Scientific, USA), followed by the addition of 7 mL of triethylamine (TEA, catalog number #81101, Merck & Co., USA). The mixture was stirred and cooled to 0°C, then 5 mL of acrylic acid (catalog number #306215, Merck & Co., USA) was added. After stirring for 12 hours, the TEA was removed by filtration, and the clear filtrate was concentrated to approximately 20 mL by vacuum rotary evaporation. The concentrate was added dropwise to 600 mL of acetone to precipitate the modified cyclodextrin, yielding AC-β-CD. Separately, 10 g of gelatin (catalog number #1288485, Merck & Co., USA) was added to 100 mL of 50°C PBS buffer, followed by the addition of 12 mL of methacrylic anhydride (catalog number #276685, Merck & Co., USA) to the 10% gelatin solution, and the mixture was stirred at 50°C for 4 hours. To remove unreacted reagents, the mixture was dialyzed against a 6 kDa molecular weight cutoff membrane (catalog number #SP132645, OriLeaf, Shanghai, China) at 45°C for one week, followed by freeze-drying at -104°C for 96 hours to obtain gelatin. Finally, the gelatin and AC-β-CD were dissolved in PBS buffer at 37°C to prepare mixed solutions with concentrations of 8% (w / v) and 10% (w / v), respectively. Finally, 0.05% (w / v) of photoinitiator (catalog number #I2959, MCE, USA) was added to prepare SupGels.

[0040] The 3D-printed scaffolds were washed sequentially with 100% ethanol and PBS solution, three times each. Naringin (catalog number #HY-N0119, MCE, New Jersey, USA) was dissolved in 1 mL of PBS to prepare a target concentration of 100 μM. SupGels were then dissolved in 1 mL of the naringin solution to prepare a naringin-SupGels mixture. After surface modification of the 3D-printed scaffolds with this mixture, they were irradiated under 365 nm UV light for 5 minutes to promote cross-linking and gelation. The treated scaffolds were labeled PHSN, while the SupGels-modified scaffolds without naringin were labeled PHS. The scaffolds were then washed three times with PBS solution to remove residual cross-linking agents. Finally, the scaffolds were lyophilized again and stored at -20°C for subsequent use.

[0041] Verification Example 1 Characterization of microporous PHSN scaffold Verify the composition, structure, and release of naringin from the PHSN scaffold in Example 1.

[0042] PHSN stent characterization: The PHSN and PHS scaffolds from Example 1 were validated. The morphology of the PNSN scaffold was characterized using scanning electron microscopy (SEM, FEI Quanta 200, Japan) combined with energy-dispersive spectroscopy (EDS), a process completed after the application of the gold coating. The chemical structure of the dried microporous scaffold was analyzed using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR, Lambda, Tianjin, China). The spectral acquisition range was 1000-4000 cm⁻¹. -1 Thirty-two scans were performed using the DiamondCrystalSmartOrbitATR attachment in Omnic 8.0 software. The cumulative release curve of naringin was determined by UV spectrophotometry. Calcium and phosphorus contents were determined using inductively coupled plasma optical emission spectrometry (ICP-OES, Agilent 7700, USA). To assess surface hydrophilicity, all scaffold samples were rinsed with deionized water, and the water contact angle of the scaffolds was measured using a scientific instrument (SCI3000F, Global Hengda Technology, Beijing, China). The degradation rate of the scaffolds (5 × 10 × 10 mm³; rotation speed: 50 rpm, 37°C) was measured and calculated on days 1, 3, 5, 7, 9, 11, 13, and 15 using the following formula: Degradation rate (%) = M t / M0×100% Where M0 is the initial support mass, M t Quality at the predetermined time point.

[0043] Naringin release behavior of the scaffold: The release behavior of naringin from the PHSN scaffold was evaluated using phosphate-buffered saline (PBS) at pH 7.5. The PHSN scaffold was placed in the release medium (PBS), and 0.5 mL of the medium was removed at predetermined time intervals (days 1, 3, 5, 7, 9, 11, 13, and 15) at 37°C and replaced with an equal volume of fresh medium to maintain a constant volume. The naringin concentration was determined by UV spectroscopy combined with analysis of the calibration curve.

[0044] result: In this invention, reference is made to Figure 2 A. After loading SupGels with naringin, both the PHS and PHSN scaffolds were attached using a hydrogel attachment method. (See reference...) Figure 2 B. Scanning electron microscopy (SEM) analysis revealed numerous pores on the surfaces of the PH, PHS, and PHSN scaffolds, indicating a microporous structure in all scaffolds. Energy dispersive spectroscopy (EDS) analysis showed that carbon (C), calcium (Ca), oxygen (O), and phosphorus (P) were uniformly distributed throughout the scaffolds. Furthermore, sodium (Na) and chlorine (Cl) were detected in the PHS scaffold. (Reference) Figure 2Further observation revealed that, in addition to Na and Cl, sulfur (S) was also present in the PHSN scaffold. This result confirms that naringin and SupGels have been successfully integrated into the surfaces of the PHS and PHSN scaffolds.

[0045] The wettability of different stents was evaluated using contact angle analysis. (Reference) Figure 2 D and Figure 2 Compared to the PH scaffold (98.14°), the contact angles of the PHS scaffold (77.63°) and PHSN scaffold (63.4°) were significantly improved. This indicates that the introduction of SupGels enhanced the hydrophilicity of the PH scaffold. Furthermore, Fourier transform infrared spectroscopy (FT-IR) analysis further revealed the chemical functional groups on the sample surface. (Reference) Figure 2 E, FT-IR spectra showed that the PHSN stent was at 1035.6, 1452.15, 1749.13, and 2933.22 cm⁻¹. -1 Characteristic absorption peaks are observed at these locations, corresponding to the vibrational modes of C=O and CH bonds, and the symmetric and asymmetric stretching vibrations of the COC bond, respectively. In contrast, naringenin shows absorption peaks at 2933.22, 1452.15, and 1035.6 cm⁻¹. -1 Characteristic peaks are observed at [values ​​missing], which are related to the asymmetric stretching vibrations of CH and COC bonds. SupGels, on the other hand, show peaks at 1749.13, 1452.15, and 1035.6 cm⁻¹. -1 Characteristic peaks were observed at the [specific locations], involving asymmetric stretching vibrations of CH, C=O, and COC bonds. Overall, these results indicate that naringenin and SupGels have been successfully integrated into the surface of the PH scaffold.

[0046] In addition, this validation example also tested the degradation characteristics of the scaffold material. Figure 2 The F-axis showed that the PHS and PHSN stents degraded significantly faster than the PH stent. (Reference) Figure 2 G. In vitro release kinetics experiments showed that naringin was rapidly released within the first 7 days, followed by a sustained slow release phase that could last up to 15 days. The degradation characteristics of the PHS and PHSN scaffolds may be related to the degradation characteristics of the SupGels surface. (Refer to...) Figure 2 H and Figure 2 I. The release curves of calcium and phosphorus were analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES). The results showed that the release of calcium and phosphorus was the highest in the PHSN scaffold and the lowest in the PH group.

[0047] Verification Example 2 In vitro biocompatibility of PHSN The biocompatibility of the PHSN scaffold in Example 1 was tested using cell culture experiments.

[0048] Cell culture: SD-BMSCs (Sprague-Dawley rat bone marrow mesenchymal stem cells) were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. (ZQY122, CELL Research, Shanghai, China). SD-BMSCs were cultured in Dulbecco modified Eagle medium (DMEM; Hyclone, USA) containing 10% fetal bovine serum (Gibco, New York, USA) and 1% penicillin (Yeasen Biotech, Shanghai, China). Human umbilical vein endothelial cells (HUVECs) were purchased from Procell Life Sciences Co., Ltd. (CL-0675; Wuhan, China) and cultured in PUMC-HUVEC-T1 dedicated growth medium (Pricella Life Sciences Co., Ltd., CM-0675). Mouse RAW264.7 cells were purchased from Procell Life Sciences Co., Ltd. (CL-0190; Wuhan, China) and cultured in DMEM (Hyclone, Utah, USA) containing 10% fetal bovine serum and 1% penicillin. All cells were cultured at 37.5°C with 5% carbon dioxide, and the culture medium was changed every three days.

[0049] Cell viability assay: To assess cell viability, this validation example included seven experimental groups with different concentrations of naringin (10, 50, 100, 150, 200, 250, and 300 μM) in microporous scaffolds. Bone marrow-derived stem cells (BMSCs) were seeded at a density of 5 × 10^4 cells / mL in 96-well plates and treated with different concentrations of naringin scaffolds. CCk-8 reagent (catalog number #C0041, Shanghai Beyotime Biotechnology Co., Ltd.) was added on days 0, 1, 2, and 3. After incubation at 37°C in the dark, absorbance was measured at 450 nm.

[0050] Scaffold toxicity to cells: To further evaluate the cytotoxicity of the scaffold, a co-culture system containing the scaffold and bone marrow mesenchymal stem cells (BMSCs) was incubated in a cell culture incubator (37°C, 5% CO2) for 24 hours to further assess the biological effects of the scaffold. Live / dead cell staining (#C2015S, Beyotime, Shanghai, China) was used for staining, and samples were observed under a confocal laser scanning microscope (CLSM, FV3000, Olympus, Tokyo, Japan). The activity of BMSCs was compared by quantifying the ratio of dead to live cells. Simultaneously, cell morphology within the scaffold was analyzed using phalloidin staining (1:1000, #PF00001, Protec, Wuhan, China).

[0051] result: Reference Figure 3B, CCK-8 assays showed that naringin effectively promoted the proliferation and differentiation of BMSCs in vitro at a concentration of 100 µM. Figure 3 A and Figure 3 C, after 1, 2, and 3 days of exposure to the extract, live / dead staining showed no cytotoxicity in any treatment group. Furthermore, referring to... Figure 3 D and Figure 3 E. Live / dead staining also revealed a small number of dead cells on PH and PHS scaffolds, while almost no dead cells were detected on PHSN scaffolds, indicating that PHSN scaffolds have no adverse effects on cells. Additionally, refer to... Figure 3 F, phalloidin staining showed that BMSCs could attach well and proliferate on all three types of scaffolds; notably, more cells attached to the PHSN scaffold compared to the PH and PHS scaffolds.

[0052] Verification Example 3 In vitro immunomodulatory effects of PHSN Verify the effect of the PHSN scaffold in Example 1 on M1 and M2 macrophages.

[0053] Inflammatory response of macrophages: The inflammatory response of macrophages was assessed using the mouse RAW264.7 macrophage cell line. First, different scaffolds were placed in the upper chamber of the Transwell system (Millipore, Bielerica, USA), and then RAW264.7 cells were seeded in the lower chamber of the Transwell system at a density of 1.5 × 10^5 cells and incubated for 48 hours. After fixation with 4% paraformaldehyde (#P0099, Beyotime, Shanghai, China), permeabilization with 0.1% Triton-X 100 (#9002-93-1, Solarbio, Beijing, China), and blocking with 3% bovine serum albumin (BSA) serum (#ST023, Beyotime, Shanghai, China), RAW264.7 cells were incubated overnight at 4°C in the dark with primary antibodies against mannose receptor (CD206, 1:300 dilution, ab300621, Abcam), inducible nitric oxide synthase (iNOS, 1:300 dilution, ab178945, Abcam), and F4 / 80 (1:300 dilution, ab6640, Abcam). Subsequently, the cells were incubated at room temperature in the dark for 1 hour with Alexa Fluor 488 goat anti-rabbit secondary antibody (1:1000 dilution, 4416s, CST) and Alexa Fluor 594 goat anti-rat secondary antibody (1:1000 dilution, 8889s, CST). After counterstaining with DAPI and mounting with anti-fluorescence quenching coverslips, the immunofluorescence signal of RAW264.7 cells was observed by confocal laser scanning microscopy, and quantitative analysis was performed using Image 1.8.0 software.

[0054] Transcriptome sequencing: To investigate the key molecular mechanisms of PHSN scaffold-mediated macrophage polarization, this validation case involved RNA sequencing of RAW264.7 cells co-cultured with the PHSN scaffold for 48 hours, with comparisons to a control group. RNA integrity was assessed using an Agilent 2100 / 4150 sequencer (Agilent Technologies, USA), ensuring all RNA integrity values ​​were above 8.0. Purified RNA was sequenced using a NovaSeq X Plus / DNBSEQ T7 sequencer (Sinovac Biotech, China), generating 150 bp strand-specific paired-end reads. Differentially expressed genes (DEGs) were screened using DESeq2 software (version 1.48.1) with a threshold of |log2 (fold change)| ≥ 1 and a corrected p-value < 0.05. Subsequently, a bioinformatics analysis platform (https: / / bioinformatics.com.cn / ) was used to generate heatmaps, volcano maps, gene ontology (GO), Kyoto Encyclopedia of Genetics and Genomes (KEGG) pathway and protein-protein interaction maps, and gene set enrichment analysis (GSEA).

[0055] result: After co-culturing RAW264.7 cells with different scaffold conditions for 48 hours, the scaffolds exhibited immunomodulatory effects. Results showed that the expression level of inducible nitric oxide synthase (iNOS) in RAW264.7 cells treated with lipopolysaccharide (LPS) was significantly upregulated, suggesting macrophage polarization towards the M1 phenotype. (Reference) Figure 4 A and Figure 4 C. Immunofluorescence staining to detect the M2 macrophage marker CD206 revealed a significantly higher number of M2 macrophages in the PHSN group compared to the PH and PHS groups. This advantage may be attributed to naringin released from SupGels coated on the scaffold surface. (Reference) Figure 4 B and Figure 4 D, In contrast, the PH, PHs, and PHSN treatment groups showed lower iNOS expression levels, indicating that these scaffolds did not induce a significant inflammatory response. (See reference...) Figure 4 F and Figure 4 Further gene expression analysis showed that the expression of M2 macrophage-related genes (CD206, Arg-1, IL-10) was upregulated, while the expression of M1 macrophage-related gene (iNOS) also showed an upregulated trend.

[0056] Verification Example 4 Angiogenesis, in vitro migration and in vitro osteogenic differentiation of PHSN Verify the angiogenesis and bone formation effects of the PHSN stent in Example 1.

[0057] Tube formation experiment: In the Transwell system, different scaffolds were placed in the upper chamber, while RAW264.7 cells were seeded in the lower chamber at a density of 1.5 × 10^5 cells. After 48 hours, the cell culture medium from the lower chamber was collected and named conditioned medium. HUVEC cells were seeded at a density of 3 × 10^4 cells in Matrigel-coated 6-well plates (catalog number #CLS354234, Corning Incorporated, USA). A 1:1 mixture of conditioned medium and DMEM was added to the 6-well plates and cultured for 12 hours. Imaging was performed using an inverted bright-field microscope (Nikon Eclipse, Tokyo, Japan).

[0058] Assessment of the migration capabilities of HUVECs: To assess cell migration rate, a scratch assay was performed as follows: Different scaffold materials were placed in the upper Transwell chamber, and 1.5 × 10^5 RAW264.7 cells were seeded in the lower Transwell chamber. After 48 hours, the lower culture medium was collected and named conditioned medium. HUVEC cells were seeded at a density of 1.5 × 10^5 cells / well in 6-well plates and cultured until 90% confluence. Straight scratches were made using a 200 µL pipette, and co-cultured for 12 hours and 24 hours, respectively. Images were taken under a microscope at the predetermined time points and analyzed using ImageJ 18.0 software.

[0059] Bone formation experiment: A differential scaffold was placed in the upper Transwell chamber, and RAW264.7 cells were seeded in the lower Transwell chamber at a density of 1.5 × 10^5 cells. After 48 hours, the cell culture medium in the lower chamber was collected and named conditioned medium. SD-BMSCs were seeded in the substrate at a density of 2 × 10^5 cells and cultured in osteogenic induction medium (RAXMX-90021, OriCell, China) for 7 and 21 days, respectively. Mineralization was then assessed on day 7 using alkaline phosphatase (ALP, P0321S, Beyotime, China) and on day 21 using alizarin red (ALIR-10001, OriCell, Suzhou, China). Images were observed under a bright-field microscope using an inverted microscope.

[0060] result: The results showed that PH, PHS, and PHSN scaffold treatments all promoted HUVEC cell migration. Figure 5 A and Figure 5 B. Notably, compared to PH and PHS, the PHSN scaffold significantly enhanced the migration ability of HUVEC cells, which is crucial for improving bone repair. (See reference...) Figure 5 C Figure 5 D and Figure 5Further experiments using the lumen formation assay confirmed that treatment with PH, PHS, and PHSN all enhanced the lumen formation ability of HUVECs. Among these, PHSN exhibited significantly superior pro-angiogenic activity compared to PH and PHS. These results indicate that PHSN can significantly promote angiogenesis and lumen formation.

[0061] Osteogenic capacity of bone marrow endothelial cells (BMECs) on days 7 and 14 was assessed using alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining. ALP staining results showed that cells in all groups exhibited varying degrees of blue-purple precipitation after treatment with PH, PHS, and PHSN. (See reference...) Figure 5 I and Figure 5 F, Notably, compared to the PH and PHS groups, the PHSN group exhibited a more significant and widespread blue-purple deposit. Furthermore, the effect of PHSN on the formation of mineralized matrix in late-stage osteogenic bone marrow stem cells (BMSCs) was examined using ARS staining. (See reference...) Figure 5 H and Figure 5 G, the results showed that the PHSN group had more and larger mineralized nodules compared to other groups. Furthermore, referring to... Figure 5 J, PHSN significantly upregulated the expression of osteogenic-related genes ALP, cyanate, and Runx2.

[0062] Verification Example 5 The role of PHSN in promoting the repair of skull defects in diabetic rats Verification of the bone defect repair effect of the PHSN scaffold in Example 1 under diabetic conditions. Stent implantation in vivo: A diabetic rat model was induced by intraperitoneal injection of 75 mg / kg streptozotocin (STZ, MCE, New Jersey, USA) after a 24-hour fast. All animal experiments were approved by Guangzhou Huateng Biopharmaceutical Technology Co., Ltd. Blood glucose levels were measured three days later; if the blood glucose level was below 13.5 mM, a second low-dose of 20 mg / kg STZ was administered. After a two-week diabetic phase, all female Wistar rats were randomly divided into four groups. Anesthesia was administered via inhalation of 2% isoflurane (#R510-22-10, RWD, Shenzhen, China). A 15 mm longitudinal scalp incision was made along the sagittal suture, and two 5 mm full-thickness defects were drilled lateral to the sagittal suture of the parietal bone. PH, PHS, and PHSN scaffolds were implanted into the defects in the experimental groups, respectively, while no implantation was performed in the control group. All rats were housed under specific pathogen-free (SPF) conditions, and skull samples were collected at 4 and 8 weeks post-implantation for subsequent analysis.

[0063] Miniature CT scan: The newly formed bone tissue at the defect site was scanned using a micro-CT scanner (micro-CT, NEMO, Jiangsu, China) with scanning parameters of 80 kV voltage, 100 μA current, and 14 μm voxel size. The acquired slices were reconstructed into three-dimensional images using accompanying software. Subsequently, three-dimensional bone morphometric analysis was performed to evaluate the regenerated bone volume, bone volume to total volume ratio (BV / TV), and bone mineral density (BMD).

[0064] HE and Masson staining: After fixation with 4% paraformaldehyde for 48 hours, the skull defect tissue was decalcified with 10% EDTA (catalog number #G1105, Servicebio, Wuhan, China) for 4 weeks. Paraffin-embedded sections with a thickness of approximately 5 μm were prepared and subsequently stained using hematoxylin-eosin (H&E, catalog number BASO, BA4025, Zhuhai, China) and Masson staining (catalog number BASO, BA4079B, Zhuhai, China), respectively.

[0065] Immunohistochemistry and immunofluorescence: Paraffin-embedded sections of skull samples were dewaxed and rehydrated sequentially. Antigen retrieval was performed by incubating the sections overnight at 58°C in sodium citrate buffer (#P0081, Beyotime, China). Subsequently, the sections were blocked with 3% bovine serum albumin (BSA). Primary antibodies against CD206 (1:200 dilution, ab300621, Abcam), inducible nitric oxide synthase (iNOS, 1:200 dilution, ab283655, Abcam), F4 / 80 (1:100 dilution, ab6640), alkaline phosphatase (ALP, 1:500 dilution, HUBBIO, PSH17-83, Hangzhou, China), cyanate (1:500 dilution, HUABIO, ER1919-20, Hangzhou, China), and CD31 (1:500 dilution, Servicebio, Wuhan, China) were incubated with the sections overnight in the dark. The sections were then incubated with the corresponding secondary antibody and DAB kit (PK1006, Proteintech, Wuhan, China). Expression changes were observed and photographed under a microscope.

[0066] result: Data analysis was performed using Origin 2024 and GraphPad Prism 10.0 software. One-way and two-way ANOVA and t-tests were used for statistical processing, and results are expressed as mean ± standard deviation. Significance levels were set as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0067] After 4 weeks of implantation, micro-CT scans showed that the PHSN group exhibited the highest regenerated bone volume, bone volume / total volume ratio (BV / TV), and bone mineral density (BMD) compared to the PH and PHS groups. Additionally, the PHS group showed better performance than the PH group in terms of bone regeneration indicators, with significant increases in regenerated bone volume, BV / TV, and BMD. Refer to Figure 6 B, Figure 6 C, Figure 6 D and Figure 6 E. After 8 weeks of implantation, the PHSN group continued to outperform the PH and PHS groups in all measurements of bone regeneration parameters. The enhanced osteogenic activity observed, especially in the naringin-containing scaffolds, confirmed the results of Verification Example 4: Naringin is an effective ingredient for promoting bone regeneration.

[0068] To further evaluate the quality of bone regeneration in the skulls of diabetic rats, histological examinations were performed using H&E staining and Masson staining techniques. Refer to Figure 6 F. H&E staining showed that after 4 weeks of treatment, only a thin layer of newly formed bone remained at the edges of the control group, while the defect area in the PHSN group was fully covered by a large amount of newly formed trabecular bone tissue, with reduced inflammatory reactions and dense formation of new capillaries. Refer to Figure 6 G. The results of Masson staining indicated that compared to the PH and PHS scaffold groups, the PHSN group showed extensive bright red mineralized bone matrix wrapped by blue osteoid tissue, suggesting more significant collagen fiber deposition in the diabetic bone defect area.

[0069] Furthermore, immunohistochemical analysis was performed to evaluate the levels of alkaline phosphatase (ALP) and osteocalcin (<cyanate>) in the bone defect area of diabetic rats to further confirm the osteogenic differentiation-promoting effect of PHSN in vivo. Refer to Figure 7 A and Figure 7 B. ALP staining at 4 and 8 weeks after implantation showed that the expression level in the PHSN group was significantly higher compared to the PHS, PH, and control groups. Refer to Figure 7 C and Figure 7 D. Similarly, <cyanate> staining at 4 and 8 weeks after implantation also indicated that the expression level in the PHSN group was significantly increased compared to the PHS, PH, and control groups. During bone remodeling, osteoblasts are responsible for bone formation, while osteoclasts are involved in bone resorption; therefore, tartrate-resistant acid phosphatase (TRAP) staining reflects the activity of osteoclasts. Refer to Figure 7 E and Figure 7 F. TRAP staining showed that at 4 weeks after implantation, the red staining in the control and PH groups was more obvious than in the PHS and PHSN groups. By 8 weeks after implantation, almost no red staining was observed in the PHSN group in TRAP staining, while some residual red staining remained in the control, PH, and PHS groups.

[0070] Verification Example 6 Further verification was conducted based on verification examples 1 to 5. Regulation of Immune System and Angiogenesis in the Body: Reference Figure 8 A and Figure 8 B. In vivo immunofluorescence staining of CD206 showed that, compared with the control group, PH group, and PHS group, the PHSN group exhibited a significantly higher CD206 expression level at 4 weeks; however, at 8 weeks, CD206 expression decreased in all treatment groups, with the PHSN group still maintaining the highest expression level. (See Figures C and...) Figure 8 Immunofluorescence staining of iNOS showed high expression levels in the control and PH groups, while the PHS and PHSN groups exhibited the lowest expression levels at both 4 and 8 weeks. Notably, iNOS expression in the PHSN group was almost undetectable. Furthermore, referring to… Figure 8 E and Figure 8 F. Tissue angiogenesis was assessed by CD31 immunofluorescence staining. Compared with the control group, PH group, and PHS group, the PHSN group showed significantly more newly formed vessels with clear lumens and boundaries at both 4 and 8 weeks.

[0071] Transcriptomic analysis of the immunomodulatory mechanism of PHSN: To investigate the immunomodulatory mechanism of PHSN, in vitro transcriptome analysis was performed using RAW264.7 cells. (Refer to...) Figure 9 A. Pearson correlation analysis showed good consistency and reliability among the groups; volcano plots and heatmaps, i.e. Figure 9 B and Figure 9 C revealed widespread differential gene expression induced by PHSN, with 272 genes upregulated and 81 genes downregulated. (See reference...) Figure 9 G, Gene Ontology (GO) database analysis showed that, compared with the control group, the PHSN group had a significant enrichment of upregulated genes related to cell motility and cellular immune regulation. Based on this, these significantly upregulated genes were divided into three main categories: macrophage polarization (…). Figure 9 D) Cell migration ( Figure 9 E) and cell differentiation ( Figure 9 F). KEGG analysis showed that the PHSN group enhanced hematopoietic lineage signaling pathways and stimulated the immune system. Furthermore, referring to... Figure 9 H, activity of the JAK-STAT, MAPK, and PI3K-AKT signaling pathways is enhanced. (See reference) Figure 9 I. Protein-protein interaction network analysis further revealed protein networks associated with the JAK-STAT, MAPK, and PI3K-AKT signaling pathways. (Refer to...) Figure 9J, GSEA analysis further highlighted the significant enrichment of the JAK-STAT signaling pathway. (Refer to...) Figure 9 K, Western blot analysis confirmed that naringin can activate the JAK-STAT signaling pathway by increasing the phosphorylation levels of JAK-2, STAT3 and STAT6.

[0072] In summary, alterations in the local microenvironment following bone defect in diabetic patients significantly impact repair outcomes. Specifically, a hyperglycemic environment not only inhibits angiogenesis but also triggers persistent inflammatory responses, severely hindering the bone defect repair process. Many traditional Chinese medicine molecules have been integrated into biomaterials to enhance their immunomodulatory and osteogenic properties. For example, the GelMa hydrogel-encapsulated potassium sodium niobate biomimetic piezoelectric scaffold loaded with icariin promotes bone defect repair by regulating macrophage polarization. 3D-printed GR scaffolds hold great potential in bone tissue engineering, acting by promoting osteoogenesis and inhibiting inflammation. 3D-printed PLGA / HA composite scaffolds demonstrate significant potential in bone tissue engineering, for example, their three-dimensional structure is conducive to cell growth and possesses good mechanical properties; however, several limitations remain in practical applications. First, the bioactivity of the PLGA / HA composite scaffold is limited by insufficient surface bioactive sites, potentially adversely affecting cell adhesion, proliferation, and differentiation. Furthermore, the hydrophobic properties of PLGA may further hinder its integration with surrounding tissues. Its inadequacy in promoting vascularization may lead to insufficient blood supply to newly formed bone tissue, ultimately affecting its long-term survival and function.

[0073] Based on Example 1 and Verification Examples 1 to 6, this invention proposes the repair effects of the traditional Chinese medicine molecule naringin and the SupGels hydrogel complex with PLGA / HA material on diabetic bone defects. Based on Verification Examples 1 to 6, naringin can promote osteoblast activity, thereby enhancing bone formation and delaying the progression of osteoporosis. Naringin can induce M2 macrophage polarization, promote osteogenic activity of bone marrow mesenchymal stem cells (BMSCs), and accelerate the healing of skull defects. Furthermore, naringin promotes the proliferation and osteogenic activity of human bone marrow stem cells (hBMSCs) by activating the ERK signaling pathway. In this invention, 100 μg / ml of naringin can promote cell proliferation. The supramolecular hydrogel (SupGels) is composed of aromatic residues of gelatin and freely diffuse, photocrosslinked acrylated β-cyclodextrin (β-CD) monomers, exhibiting excellent biological activity.

[0074] Referring to Validation Example 1, in this invention, naringin and SupGels successfully anchored on the surface of the PLGA / HA scaffold, allowing for the slow release of naringin from the scaffold. Furthermore, this PHSN scaffold exhibits degradation properties, releasing calcium (Ca) and phosphorus (P) ions, and demonstrates excellent hydrophilicity. Referring to Validation Example 3, the optimal concentration of naringin was 100 μM, which promoted subsequent cell proliferation. More importantly, the application of SupGels significantly improved the cell recruitment performance and cell viability of the PHSN scaffold. These results fully demonstrate the biocompatibility of this scaffold.

[0075] Bone repair after injury is an inflammatory response involving multiple cellular and molecular activities, in which macrophages play a key role. In the early inflammatory phase of bone injury healing, macrophages typically polarize to a pro-inflammatory M1 phenotype via the classical pathway. This phenotypic shift enables macrophages to produce a range of inflammatory cytokines, including TNF-α, IL-6, and IL-1β, which can effectively recruit various immune cells and support the clearance of damaged tissues and harmful substances

[41] . Regulating the shift of macrophage phenotype from M1 to M2 through precise temporal control in the later stages of regeneration is a key issue in promoting bone repair. Referring to validation example 3, PHSN and 264.7 cell treatments can effectively regulate macrophage polarization to the M2 phenotype by upregulating the gene expression levels of CD206, Arg-1, and IL-10; conversely, this treatment inhibits the polarization of M1 macrophages by downregulating the gene expression levels of iNOS, IL-6, and IL-23. Therefore, these results indicate that the PHSN scaffold can modulate macrophage polarization toward the M2 phenotype, laying the foundation for subsequent bone repair.

[0076] Angiogenesis and osteogenesis are two key processes in the repair of diabetic bone defects, and their coupling effect is of decisive significance. During normal bone repair, angiogenesis and osteogenesis are interdependent; angiogenesis not only provides essential oxygen and nutrients for bone tissue, but also promotes osteoblast differentiation and bone formation by secreting various growth factors such as VEGF and BMP2. However, under diabetic conditions, hyperglycemia and oxidative stress disrupt this coupling relationship, leading to reduced angiogenesis and impaired bone formation. Referring to Verification Example 4, PH, PHS, and PHSN scaffolds were co-cultured with macrophages, and the conditioned medium was collected to evaluate the migration and tube formation ability of HUVECs, as well as the osteogenic differentiation ability of SD-BMSCs. The results showed that the conditioned medium of the PHSN scaffold significantly promoted the migration and tube formation of HUVECs. In addition, it enhanced the osteogenic differentiation of BMSCs by upregulating the expression of alkaline phosphatase (ALP), osteocalcin (<cyanate>), and Runt-related transcription factor 2 (Runx2) genes. Referring to Verification Example 5, in vivo experiments, micro-computed tomography (micro-CT), hematoxylin-eosin (HE) staining, and Masson staining showed that PHSN exhibited excellent osteogenic repair effects. Analysis of osteogenesis-related markers showed that the PHSN group exhibited the highest positive expression levels of ALP and <cyanate>, further confirming its efficacy in bone repair. TRAP staining showed that PHSN could inhibit osteoclast formation. In addition, referring to Verification Example 6, staining of macrophage polarization markers showed that PHSN could regulate the polarization of macrophages towards the M2 phenotype while inhibiting the formation of M1 macrophages. CD31 staining showed that PHSN could promote angiogenesis, which may be attributed to the application of SupGels and naringin. These results are consistent with the cell experiment data, jointly demonstrating that PHSN can promote angiogenesis and accelerate bone repair by regulating macrophage polarization.

[0077] To further investigate the potential mechanism by which naringin regulates macrophage polarization, referring to Example 6, this invention employed RNA sequencing (RNA-Seq) technology to analyze gene expression changes in macrophages from the PHSN group and the control group. Principal component analysis (PCA) was used to differentiate gene expression patterns between the control and PHSN groups. A total of 353 differentially expressed genes were identified in both the PHSN scaffold-treated and control groups, with 272 genes upregulated and 81 genes downregulated. Gene ontology enrichment analysis showed that gene expression changes in the PHSN group were correlated with receptor ligand activity, cytokine activity, and growth factor activity. Macrophage phenotype and cell migration play a crucial role in the regulation of bone defect repair. The results showed that upregulated genes were associated with M2-type macrophages, cell migration, and tissue repair, indicating that the PHSN scaffold can promote macrophage differentiation into the M2 type and enhance cell migration and tissue repair capabilities. Furthermore, KEGG enrichment analysis revealed that the JAK / STAT signaling pathway is closely related to macrophage polarization and cell migration.

[0078] The JAK / STAT signaling pathway plays a crucial role in regulating macrophages. For example, Sr-PTL-MCM can promote M2 macrophage polarization through the JAK-STAT and MAPK signaling pathways, thereby enhancing osteogenic differentiation of mesenchymal stem cells (MSCs). The Met@ZIF-8@PDA micron-scale scaffold can increase the M2 / M1 ratio and promote osteogenic differentiation of damaged MSCs. Concentrated growth factor (CGF) induces M2 macrophage polarization by promoting STAT3 phosphorylation, suggesting that this process may be related to the STAT3 signaling pathway. To verify the role of these signaling pathways, this invention used Western blotting to detect the protein expression levels of JAK-2, phosphorylated JAK-2, STAT3, phosphorylated STAT3, STAT6, and phosphorylated STAT6. JAK-2 is a cytoplasmic protein tyrosine kinase that regulates the expression of downstream STAT genes. Increased levels of phosphorylated JAK-2 are closely related to increased levels of downstream p-STAT3; for example, C4BPA can promote JAK-2 phosphorylation and enhance STAT3 phosphorylation. Using JAK2 inhibitors or knocking down JAK2 via siRNA significantly reduced p-STAT3 levels. Furthermore, STAT6 activation is associated with M2 microglia polarization, and IL-4 may partially promote M2 microglia / macrophage polarization through the JAK1 / STAT6 pathway.

[0079] In summary, the PHSN scaffold proposed in this invention has shown significant effects on regulating macrophage polarization in both in vitro and in vivo experiments, and can effectively promote the repair of bone defects in the context of diabetes.

[0080] The above description is merely an exemplary embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention specification and drawings under the technical concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A composite scaffold for promoting bone regeneration in diabetic patients, characterized in that, include Three-dimensional porous framework; Bioactive hydrogel layer loaded with naringin; The bioactive hydrogel layer loaded with naringin is coated on the surface and inner walls of the pores of the three-dimensional porous framework.

2. The composite scaffold for promoting bone regeneration in diabetic patients as described in claim 1, characterized in that, The three-dimensional porous framework is composed of polylactic acid-glycolic acid copolymer and hydroxyapatite.

3. The composite scaffold for promoting bone regeneration in diabetic patients as described in claim 2, characterized in that, The mass ratio of hydroxyapatite to polylactic acid-glycolic acid copolymer is 1:

4.

4. The composite scaffold for promoting bone regeneration in diabetic patients as described in any one of claims 1 to 3, characterized in that, The bioactive hydrogel layer is a supramolecular hydrogel, which is formed by cross-linking gelatin and acrylic β-cyclodextrin under ultraviolet light.

5. The composite scaffold for promoting bone regeneration in diabetic patients as described in claim 4, characterized in that, In the supramolecular hydrogel, the concentration of gelatin is 8% (w / v), and the concentration of acrylic β-cyclodextrin is 10% (w / v).

6. The composite scaffold for promoting bone regeneration in diabetic patients as described in claim 4, characterized in that, The loading concentration of naringin in the bioactive hydrogel layer is 100 μM.

7. The composite scaffold for promoting bone regeneration in diabetic patients as described in claim 4, characterized in that, The three-dimensional porous skeleton was made by 3D printing.

8. A method for preparing a composite scaffold that promotes bone regeneration in diabetic patients, characterized in that, Includes the following steps: S1. Prepare a three-dimensional porous framework; S2. Preparation of bioactive hydrogel loaded with naringin; S3. The bioactive hydrogel loaded with naringin is coated on the surface of the three-dimensional porous framework and cross-linked by ultraviolet light irradiation to obtain the composite scaffold.

9. The method for preparing a composite scaffold for promoting bone regeneration in diabetic patients as described in claim 8, characterized in that, The specific steps of S1 are as follows: Hydroxyapatite and polylactic acid-glycolic acid copolymer were mixed at a mass ratio of 1:4 and dissolved in 1,4-dioxane to prepare a 10% (w / v) homogeneous slurry. The slurry was then 3D printed and freeze-dried to obtain the three-dimensional porous skeleton.

10. The composite scaffold for promoting the regeneration of diabetic bone defects as described in any one of claims 1-7, used in the preparation of a medicament or medical device for treating diabetic bone defects.

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