Immune metabolism regulation unblocking type gel scaffold and preparation method and application thereof

Abstract

This invention discloses an immunometabolic regulation unsealing gel scaffold, its preparation method, and its application. The scaffold has a core-sheath structure; the core layer of the core-sheath structure contains methacryloyl hyaluronic acid, lithium magnesium silicate, stromal cell-derived factor-1α, and a photoinitiator; the sheath layer of the core-sheath structure contains puerarin and zinc ions. The immunometabolic regulation unsealing gel scaffold provided by this invention delivers PUE / Zn via programmed delivery. 2+ With SDF-1α / Mg 2+ This scaffold effectively couples macrophage arginine metabolism reprogramming with stem cell recruitment and osteogenic activation. It can effectively unblock the blocked Arg-1 / ODC pathway in macrophages within the osteoporotic microenvironment and activate the osteogenic program of BMSCs through putrescine-mediated transcellular metabolic transport, providing a novel strategy for repairing osteoporotic bone defects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an immune metabolism regulation and unsealing gel scaffold, its preparation method, and its application. Background Technology

[0002] Osteoporosis (OP)-related bone defects pose a significant challenge to bone regeneration in the context of aging, with repair failure often manifesting as delayed bone healing, inadequate osseointegration, and poor regeneration quality. Although anti-resorption therapies, osteopromoting drugs, and bone replacement materials can improve bone metabolism or provide structural support to some extent, their overall efficacy in repairing defects in the context of osteoporosis remains limited. This suggests that OP-related bone regeneration disorders are not solely due to decreased osteogenic capacity, but more likely involve long-term imbalances in the local regenerative microenvironment, leading to impaired repair initiation and a disconnect in regenerative signal transmission.

[0003] In the early stages of bone defect repair, the local immune microenvironment determines whether subsequent tissue regeneration can be successfully initiated. Macrophages are not only the core regulators of the inflammatory response but also crucial hubs connecting immune regulation and osteogenic processes. Recent studies have shown that the arginine metabolic shunting status of macrophages directly affects their functional phenotype and their shaping of the bone regeneration microenvironment: when arginine enters the iNOS pathway, it often maintains amplified inflammation and tissue damage; while when it shifts to the ODC axis, it can promote the production of repair metabolites such as polyamines, providing metabolic support for matrix reconstruction and osteogenic initiation. However, in the OP-related microenvironment, persistent inflammation and metabolic disturbances often restrict this pro-repair shunting, making it difficult for macrophages to establish metabolic output programs that support bone regeneration. Therefore, overcoming the bottleneck in macrophage arginine metabolism and further reprogramming its metabolism into osteogenic signals is key to breaking through the bottleneck in OP bone defect repair.

[0004] It is worth noting that bone regeneration is essentially a dynamic process with distinct stages. In the early stages of repair, it is crucial to prioritize correcting the imbalanced immune-metabolic state to create conditions for regeneration initiation. Subsequently, it is necessary to recruit and anchor osteogenic-related cells to receive the pro-repair signals established in the earlier stages and drive new bone formation. In other words, truly effective intervention should not be limited to a single anti-inflammatory or osteogenic approach, but should achieve continuous regulation from early microenvironmental correction to subsequent osteogenic activation. However, existing bone repair materials mostly focus on single functional outputs, making it difficult to match the inherent need for a continuous transition from early immune-metabolic remodeling to subsequent osteogenic activation in OP bone defect repair. Therefore, constructing material systems that can adapt to different repair stages and achieve programmed functional switching may provide a new strategy for OP bone defect regeneration. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing an immune metabolism regulation unsealing gel scaffold, its preparation method, and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect is to provide an immune metabolic regulation and unsealing gel scaffold, the scaffold having a core-sheath structure; The core layer of the core-sheath structure comprises methacrylamide hyaluronic acid, lithium magnesium silicate, stromal cell-derived factor 1 alpha (SDF-1α), and a photoinitiator; The sheath layer of the core-sheath structure contains puerarin and zinc ions.

[0007] Furthermore, the photoinitiator is I2959.

[0008] The second aspect is to provide a method for preparing the above-mentioned immunometabolic regulation unsealing gel scaffold, including the following steps: Step 1: Methacrylamide hyaluronic acid, lithium magnesium silicate, matrix cell-derived factor-1α and photoinitiator are dissolved in deionized water to prepare the core layer bio-ink. Step 2: Disperse puerarin in deionized water, heat to dissolve, cool down, add zinc salt, stir evenly, and prepare sheath bio-ink. Step 3: Load the core layer bio-ink from Step 1 and the sheath layer bio-ink from Step 2 into the inner and outer cylinders of the coaxial 3D printer, respectively, and print using the coaxial nozzle. Step four: The scaffold printed in step three is subjected to photocuring and cross-linking to obtain a gel scaffold with a core-sheath structure.

[0009] Furthermore, in the core layer bio-ink, the concentration of methacrylamide hyaluronic acid is 1-4 wt%, the concentration of lithium magnesium silicate is 2-8 wt%, and the concentration of matrix cell-derived factor-1α is 1×10⁻⁶. -5 -1×10 -4 The concentration of photoinitiator is 0.1–0.5 wt%; in the sheath bioink, the concentration of puerarin is 1–10 wt%, and the concentration of zinc salt is 0.5–2 wt%.

[0010] Furthermore, in the core layer bio-ink, the concentration of methacrylamide hyaluronic acid is 4 wt%, the concentration of lithium magnesium silicate is 4 wt%, and the concentration of matrix cell-derived factor-1α is 5 × 10⁻⁶. -5 The concentration of photoinitiator is 0.2 wt%; the concentration of puerarin in the sheath bioink is 4 wt%, and the zinc salt is ZnSO4·7H2O with a concentration of 1 wt%.

[0011] Furthermore, in step two, the heating and dissolution conditions are as follows: heating at 95–100 °C for 5–15 minutes; adding zinc salt when the temperature is lowered to 30–40 °C.

[0012] Furthermore, in step three, the printing speed is 400–700 mm / s, and the platform temperature is -10–0 °C; the mixing ratio of the core layer bio-ink to the sheath layer bio-ink is 6:4.

[0013] Furthermore, in step four, the wavelength of the light source for photocuring crosslinking is 405 nm, and the irradiation time is 0.5–3 min.

[0014] The third aspect is to provide the application of the above-mentioned immune metabolic regulation unsealing gel scaffold in the preparation of products for the repair of osteoporotic bone defects.

[0015] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: The immune metabolism-regulating desealing gel scaffold provided by this invention delivers PUE / Zn via programmed delivery. 2+ With SDF-1α / Mg 2+ This scaffold effectively couples macrophage arginine metabolism reprogramming with stem cell recruitment and osteogenic activation. It can effectively unblock the blocked Arg-1 / ODC pathway in macrophages within the osteoporotic microenvironment and activate the osteogenic program of rat bone marrow mesenchymal stem cells (BMSCs) through putrescine-mediated transcellular metabolic transport, providing a novel strategy for repairing osteoporotic bone defects. Attached Figure Description

[0016] Figure 1 The following are the physicochemical characterization results of the HP hydrogel scaffold; where A represents the microstructure; B represents the elemental distribution; C represents the core-sheath microstructure; D represents the swelling photograph; E represents the swelling ratio curve; and F represents... 1 H-NMR plot; G represents rheological properties; H represents stress-strain curve; I represents compressive modulus.

[0017] Figure 2The effects of HP hydrogel scaffolds on the proliferation, antioxidant stress resistance, and polarization capacity of RAW264.7 cells were shown. A represents the proliferation of RAW264.7 cells after culturing in HP hydrogels (extraction solutions) with different PUE concentrations; B represents the fluorescence staining results of the antioxidant stress resistance of each group of cells; C represents the quantitative results of reverse transcription quantitative polymerase chain reaction (RT-qPCR) for Arg-1, YM-1, IL-1β, and iNOS; D represents the protein expression levels of Arg-1 and iNOS; and E represents the protein expression distribution of Arg-1 and iNOS.

[0018] Figure 3 The HP hydrogel scaffold was shown to recruit bone marrow mesenchymal stem cells and promote osteogenic differentiation. In the diagram, A shows ALP staining; B shows ARS staining; C shows quantitative results of ALP and ARS staining; D shows protein expression levels of Col-1α and OCN; E shows the protein expression distribution of Col-1α and OCN; F shows the cytoskeleton of BMSCs recruited by the material; G shows the spreading state of BMSCs on the material; H shows the RT-qPCR quantitative results of ALP, Runx2, and BMP2 after 7 and 14 days of culture; and I shows the Western blot analysis of OPN, OCN, and Col-1α.

[0019] Figure 4 The study demonstrated the ability of the HP hydrogel scaffold to promote vascularization; where A represents the horizontal migration ability of HUVECs; B represents the vascular structure; C represents the vertical migration ability of HUVECs; D represents the migration rate, number of vascular nodules, number of vascular branches, total vascular length, and number of migrating cells of HUVECs; E represents the protein expression distribution of CD31; and F represents the protein expression level of CD31.

[0020] Figure 5 The study demonstrates the repair effect of the HP hydrogel scaffold in a rat model of osteoporosis. A shows the Micro-CT and 3D reconstruction of the femur; B shows BMD, BV / TV, Tb.N, and Tb.Sp; C shows the hematoxylin and eosin (HE) staining and Masson's trichrome (Masson) staining of the femur; and D shows the HE staining of the heart, liver, spleen, lungs, and kidneys.

[0021] Figure 6 Immunofluorescence and immunohistochemical staining images of the femur of an osteoporotic rat model repaired with an HP hydrogel scaffold; where A represents CD31; B represents OCN; C represents iNOS and Arg-1; and D represents Col-1α.

[0022] Figure 7 The results show the transcription of genes related to lysosomal remodeling and amino acid transport driven by the HP hydrogel scaffold in RAW 264.7 cells. A represents PCA analysis; B is a volcano plot; C is a heatmap related to lysosomal remodeling and amino acid transport; D shows the relative abundance of lysosomal remodeling-related genes p62, ATP6V0A1, and amino acid transport-related gene SLC7A14; EF represents GO enrichment analysis; GH represents KEGG enrichment analysis; I represents GSEA enrichment analysis; and K represents Western blotting analysis of lysosomal remodeling-related proteins LAMP1, LC3-Ⅰ, LC3-Ⅱ, p62, ATP6V0A1, and amino acid transport-related protein SLC7A14.

[0023] Figure 8 The results show that the HP hydrogel scaffold generates putrescine through the Arg-1 / ODC metabolic pathway in RAW 264.7 cells, activating the mTOR pathway in BMSCs. In the figures, A is PCA analysis; B is a volcano plot; C is a bubble scatter plot; D is GO enrichment analysis; E is KEGG enrichment analysis; F is a heatmap related to arginine metabolism; G is the relative abundance of arginine metabolites Arginine, Ornithine, and Putrescine; H is the Western blot analysis of key enzymes in the arginine metabolic pathway shunting, iNOS, ODC, and Arg-1; I is the RT-qPCR quantification of OPN and Col-1α; and J is the Western blot analysis of mTOR pathway-related proteins p-mTOR, mTOR, p-S6, and S6. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0025] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions or as recommended by the manufacturer. Unless otherwise stated, all reagents and materials used in the following examples were commercially available. The main reagents are as follows: 2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylphenylacetone (I2959, Sigma-Aldrich, Germany), lithium magnesium silicate (Lap, BYK-Chemie GmbH, Germany), methacrylic anhydride (Aladdin Biochemical Technology Co., Ltd., China), puerarin (PUE, Aladdin Biochemical Technology Co., Ltd., China), hyaluronic acid (HA, Bloomage Biotechnology Co., Ltd., China); stromal cell-derived factor-1α (SDF-1α, Beyotime Biotechnology, China), zinc sulfate heptahydrate (Aladdin Biochemical Technology Co., Ltd., China), phosphate-buffered saline (PBS, Wuhan ServicebioTechnology Co., Ltd., China), and anhydrous ethanol (Titan Scientific Co., Ltd., China). All reagents were stored according to the supplier's recommended conditions and were not further purified before use.

[0026] Example 1 1.1 Synthesis of Methacrylamide Hyaluronic Acid (HAMA) 3.3 g of hyaluronic acid was dissolved in 500 mL of deionized water under ice bath conditions to form a homogeneous solution. Then, 20 mL of methacrylic anhydride solution was slowly added dropwise. The pH of the reaction system was adjusted to 8-9 with 5M NaOH aqueous solution, and the reaction was continued under ice bath conditions for 24 h. After the reaction was completed, the reaction solution was transferred to a dialysis bag (MWCO 8000-14000 Da), and the deionized water was dialyzed for 72 h, with the dialysate being changed periodically to ensure complete removal of unreacted methacrylic anhydride and byproducts. The dialysis solution was freeze-dried to obtain white HAMA powder. The obtained product was processed... 1 H NMR (BRUKER AVANCE 400, Bruker Corporation, Germany) identification.

[0027] 1.2 Fabrication of HAMA / Lap / SDF-1α@PUE / Zn(HP) scaffolds by 3D printing The lyophilized HAMA powder was dissolved in deionized water to prepare a 1-4 wt% prepolymer solution, and 0.2 wt% I2959, 2-8 wt% Lap, and 5×10-5 HAMA / Lap / SDF-1α (HLS) bio-ink was obtained by fully dissolving wt% SDF-1α.

[0028] Puerarin (PUE) was dispersed in deionized water at different mass concentrations (0 wt%, 1 wt%, 4 wt%, 6 wt%, 10 wt%). After magnetic stirring for 30 min, it was heated at 100 °C for 10 min to completely dissolve it. While stirring continuously, the temperature was lowered to 37 °C and 1 wt% ZnSO4·7H2O was added. After stirring evenly, PUE / Zn (PZn) bio-ink was obtained.

[0029] After optimization, the preferred core layer ink composition of this invention is 4 wt% HAMA, 4 wt% Lap, and 5×10 -5 The sheath ink composition is 4 wt% SDF-1α and 0.2 wt% I2959, and 1 wt% PUE and 1 wt% ZnSO4·7H2O. However, the above concentrations are only illustrative and can be adjusted within a certain range as needed by those skilled in the art.

[0030] The two bio-inks were transferred into the printing barrel of an extrusion-type bio-3D printer (EFL-BP-6602P, EngineeringForLife (EFL) Co., Ltd., China) and coaxially printed at a printing speed of 500 mm / s, a platform temperature of -5 °C, and an inner-outer layer mixing ratio of 6:4. Immediately after printing, the scaffold was cured by irradiation with a 405 nm light source for 1 min to ensure complete cross-linking, resulting in an HP scaffold with an inner layer of HLS and an outer layer of PZn. Control scaffolds (HAMA scaffold, PZn scaffold, and HLS scaffold) were prepared using a single-nozzle printing method.

[0031] 1.3 Material Characterization After complete freeze-drying and gold sputtering, the scaffold's surface morphology and pore structure were observed using a scanning electron microscope (SEM, SU8100, Hitachi, Japan), and energy dispersive spectroscopy (EDS) was performed to determine the spatial distribution of elements. To verify the integrity of the inner and outer layer interfaces, 0.5 wt% of red or green fluorescent protein microfilaments were added to the inks of the inner and outer layers, respectively, followed by 3D printing. Finally, the coaxial structure was observed using an inverted fluorescence microscope (TS2-FL, Nikon Instruments Co., Ltd., China). The scaffold's swelling performance was evaluated by immersing the scaffold in PBS, weighing its wet weight at preset time points, and calculating the swelling ratio. Rheological properties were tested using a rotational rheometer (Kinexus ultra+, NETZSCH Analyzing & Testing, Germany), and the storage modulus (G′) and loss modulus (G″) were recorded to evaluate the mechanical rheological characteristics of the materials. Compressive properties were tested using a universal testing machine (AGS-X, Shimadzu, Japan), and the compressive modulus and stress-strain curves were obtained using a loading rate of 1 mm / min.

[0032] 1.4 Characterization Results SEM images show that the HP scaffold has a uniform pore distribution, ensuring good cell adhesion and in-situ colonization. The porosity of the HLS scaffold is lower than that of the HAMA scaffold, possibly because the addition of Lap significantly increases the ink viscosity, making it less likely for the system to form a large pore structure during printing and crosslinking. The porosity of the HP scaffold is higher than that of the HLS scaffold, which may be related to the larger intrinsic pore size of the gel network formed by PUE, resulting in a more loose and porous macrostructure after molding. Figure 1 (A). EDS analysis showed that Lap and Zn 2+ Evenly load on the support surface ( Figure 1 (B) Figure 1 The middle C visually demonstrates the well-organized coaxial structure of the HP support, with its inner and outer layers exhibiting a typical "core-sheath" coaxial arrangement and a clear and continuous interface. This indicates that during the printing process, the coaxial extruder can stably achieve synchronous deposition of the inner and outer phase inks, thereby constructing a complete and uniform three-dimensional coaxial support.

[0033] All three scaffolds reached swelling equilibrium within 30 minutes, but their swelling performance showed significant differences: the HAMA scaffold had the highest equilibrium swelling rate, followed by the HLS scaffold, and the HP scaffold had the lowest. This trend may be closely related to differences in network structure and composition: the pure HAMA scaffold has high hydrophilicity and relatively low network density, enabling it to adsorb and hold more water; the HLS scaffold, due to the introduction of Lap, partially densifies the gel network, thus limiting water entry to some extent, resulting in a slightly lower swelling capacity than HAMA; while the HP scaffold, with the addition of PUE, has a more compact and stable gel network, reducing free volume and effective hydrophilic sites, leading to the lowest overall swelling degree. Figure 1 middle DE).

[0034] 1 In 1H NMR, a methyl (-CH3) signal peak of -NHCOCH3 is visible at 1.8 ppm, and a pair of characteristic peaks belonging to the allyl proton (-CH2=) at the methacrylic acid group terminal appear at 5.6 ppm and 6.1 ppm. Figure 1 The above results together confirm the successful grafting of methacryloyl groups onto HA.

[0035] Rheological test results show that the storage modulus (G′) and loss modulus (G″) of the HP system are significantly higher than those of HLS and PZn, and G′ is always greater than G″. Figure 1 The high G′ and G″ values ​​indicate that HP inks possess a stronger elastic network structure and better shape retention. HLS has medium G′ and G″ values, while PZn has the lowest values ​​for both. Considering the 3D printing process, higher G′ and appropriate G″ facilitate smooth ink extrusion under nozzle shear and rapid recovery of structure after deposition, maintaining the contour of printed lines and interlayer stacking stability. Therefore, HP inks exhibit the best printability.

[0036] Compression mechanical test results show that HP has a compression modulus of 52±14 kPa, which is 2.2 times that of HLS and 5.6 times that of PZn. Figure 1 (Middle HI). This result indicates that the HP stent possesses the most stable three-dimensional network structure. Sheath PUE and Zn 2+ The formation of metallic coordination bonds enhances the stability of the system, while the core layer, through the double-charge characteristics and high specific surface area of ​​Laponite, achieves electrostatic self-assembly and dissociation release with SDF-1α. Both rely on HAMA's -COO -Electrostatic forces are formed with the negative charge of SDF-1α. This helps maintain the effective concentration of SDF-1α in the core layer, thereby achieving continuous stem cell homing and reducing systemic side effects. In the context of the 3D printing process, a higher compressive modulus reflects tighter interlayer bonding and more complete and continuous pore walls after printing, which is beneficial for maintaining the intended macroscopic morphology and pore structure in subsequent operations, and is less prone to collapse or excessive deformation. In contrast, HLS and PZn scaffolds, due to their lower overall modulus, are more prone to structural deformation under pressure or immersion expansion.

[0037] Compared to conventional HAMA-based 3D-printed scaffolds, the HP scaffold of this invention exhibits a superior pore structure. Its pore size is approximately 884±86 μm, which facilitates the exchange of cellular nutrients and the removal of waste products, promoting osteogenic growth. Simultaneously, the equilibrium swelling ratio of the HP scaffold is significantly lower than that of most previously reported HAMA-based hydrogels, indicating higher structural stability, less change in pore size and shape in body fluid environments, and stronger support. The compressive modulus of HP (52±14 kPa) is comparable to that of high-performance HAMA composite hydrogels, demonstrating excellent mechanical properties.

[0038] Example 2 2.1 Experimental Methods 2.1.1 Cell culture and preparation of conditioned medium Mouse RAW 264.7 cells and human umbilical vein endothelial cells (HUVECs) were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10 wt% fetal bovine serum (FBS) and 1 wt% penicillin / streptomycin (P / S). BMSCs were cultured in α-modified minimum essential medium (α-MEM) containing 10 wt% FBS and 1 wt% P / S. All cells were incubated at 37 °C in a 5% CO2 incubator to maintain proliferation. For cell observation experiments on the material surface, the scaffold was first soaked in 75% ethanol, washed with PBS, and sterilized by ultraviolet (UV) irradiation. Cells were then seeded onto the scaffold surface and incubated statically to complete cell attachment. In the cell observation experiment culturing in the extract, the material was first soaked in the culture medium for 24 h to obtain the material extract, and then the cells were cultured in it.

[0039] To obtain conditioned medium (CM) for macrophages, RAW 264.7 cells were grown to 70-80% confluence under standard culture conditions, and then induced for 3 days with either compound or material extract. After induction, the culture medium was discarded, and the cells were gently washed with pre-warmed PBS, followed by incubation with fresh culture medium for 24 h. The supernatant was then carefully collected and centrifuged at 300 × g for 5 min to remove suspended cells and debris. The supernatant was then filtered through a 0.22 μm polyethersulfone (PES) filter membrane, and the resulting clear, cell-free supernatant was used as the conditioned medium. All CM was immediately aliquoted and stored at −80 °C to avoid loss of viability due to repeated freeze-thaw cycles.

[0040] 2.1.2 Cell proliferation capacity RAW 264.7 cells were cultured in HP scaffold extract containing 0.1–1.0 wt% PUE (labeled PUE 0.1, PUE 0.3, PUE 0.6, and PUE 1.0, respectively), and cell proliferation was assessed using a Cell Counting Kit-8 (CCK-8). Live and dead cells were then stained using a Calcein / PI cell viability and cytotoxicity assay kit, and images were acquired under an inverted fluorescence microscope.

[0041] 2.1.3 Reactive oxygen species (ROS) scavenging capacity The ROS scavenging ability of the material was detected using a reactive oxygen species (ROS) detection kit. RAW 264.7 cells were cultured in the material extract, the culture medium was discarded, and 0.1 wt% 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and 100 μM H2O2 were added. After 2 h, the fluorescence intensity was observed under an inverted fluorescence microscope.

[0042] 2.1.4 Macrophage polarization ability RAW 264.7 cells were cultured in the material extract and then subjected to immunofluorescence analysis. iNOS was used as the M1 marker and Arg-1 as the M2 marker. After fixation, permeabilization, and blocking, the cells were sequentially incubated with primary and secondary fluorescent antibodies. Images were then acquired using a laser confocal microscope (Sp8, Leica Microsystems Trading Co., Ltd., China), and fluorescence intensity was quantified using ImageJ. To analyze trends at the gene level, total RNA was extracted from the cells and reverse transcribed into cDNA. The expression of polarization (iNOS, YM-1, and Arg-1) and inflammation-related genes (IL-1β) was measured using RT-qPCR, and the results were normalized using the ΔΔCt method.

[0043] 2.1.5 In vitro osteogenic differentiation capacity After inducing and culturing BMSCs in CM, alkaline phosphatase (ALP) staining was performed on day 7 using the BCIP / NBT alkaline phosphatase colorimetric kit, and ALP activity was quantified using an alkaline phosphatase assay kit. On day 14, Alizarin Red S staining was performed to analyze calcium salt deposition, and absorbance was measured using cetylpyridinium chloride destaining solution for quantification. To further confirm the expression of osteogenic marker proteins, Col-1α and OCN were immunofluorescence stained, observed using laser confocal microscopy, and quantified using ImageJ. F-actin staining was used to observe cell spreading, and SEM was used to analyze the adhesion characteristics of the cell-material interface. RT-qPCR was used to detect changes in Runx2, ALP, and BMP2 gene expression to assess the osteogenic induction effect at the gene level. Western blot (WB) was used to further validate the protein expression levels of OPN, OCN, and Col-1α.

[0044] 2.1.6 In vitro angiogenesis capacity HUVECs were cultured in CM and subjected to a scratch assay to analyze their horizontal migration ability. Scratch width changes were observed at 0 h and 6 h, and migration rates were calculated. In the tube formation assay, HUVECs were seeded on a Matrigel surface, CM was added, and tube formation was observed at 6 h. Quantification was performed using the number of nodes, branches, and total tube length. The Transwell migration assay was used to assess vertical migration ability; HUVECs were cultured in CM for 24 h, and the number of migrating cells was observed and counted. Differences in CD31 expression in HUVECs cultured in different CMs were detected by immunofluorescence staining, and quantification was performed using ImageJ.

[0045] 2.1.7 Establishment of an osteoporotic rat model and in vivo assessment of bone repair Osteoporosis was established in female SD rats via bilateral ovariectomy. Eight weeks post-surgery, standard round bone defects (3 mm in diameter and 3 mm in depth) were prepared in the femur and scaffolds of different materials were implanted. Tissue samples were harvested at predetermined post-operative times. A three-dimensional model was constructed using Micro-CT, and bone tissue parameters such as BMD, BV / TV, Tb.N, and Tb.Sp were calculated to assess new bone formation. Tissue repair was analyzed using HE and Masson staining, and differential expression of markers CD31, OCN, iNOS, Arg-1, and Col-1α was analyzed using immunofluorescence or immunohistochemistry. The biocompatibility of the materials was assessed by HE staining of major organs (heart, liver, spleen, lung, and kidney).

[0046] 2.1.8 Transcriptome sequencing and metabolomics analysis RAW 264.7 cells were cultured in standard culture medium and HP extract, designated as the Control group and HP group, respectively. RNA was extracted from these two groups of cells for high-throughput sequencing, differentially expressed genes were analyzed, and GO, KEGG, and GSEA enrichment analyses were performed. Metabolomics analysis was conducted using the LC-MS / MS platform (Q Exactive Plus, Thermo Fisher Scientific Inc., USA), including peak extraction, alignment, metabolite identification, and metabolic pattern difference analysis for all samples. The threshold for statistically significant differential expression was set as P < 0.05 and |log2(FoldChange)| > 1.

[0047] Western blotting was used to determine the expression levels of proteins related to the mechanistic pathway. Bafilomycin A1 (BafA1), an autophagy inhibitor, was used to investigate the effect of HP on RAW. 264.7 Effects of HP on the expression levels of lysosomal amino acid transport and autophagy-related proteins (LAMP1, ATP6V0A1, SLC7A14, p62, LC3-Ⅰ, and LC3-Ⅱ); The effects of HP on the expression levels of arginine metabolism shunting-related proteins (iNOS, ODC, and Arg-1) were investigated using lipopolysaccharide (LPS) as an M1 inducer and interleukin-1 / 10 / 13 (IL-1 / 10 / 13) as an M2 inducer. The effects of HP on the expression levels of osteogenic differentiation signaling pathway-related proteins (p-mTOR, mTOR, p-S6, and S6) in BMSCs were investigated using conditioned medium supplemented with Nω-hydroxy-nor-L-arginine (nor-NOHA) and L-arginine (L-Arg).

[0048] By combining metabolomics, transcriptomics, and Western blot data to construct regulatory networks, we can reveal the potential mechanisms by which materials regulate metabolism, immunity, and osteogenic processes from multiple dimensions.

[0049] 2.1.9 Statistical Analysis All quantitative data were analyzed using GraphPad Prism 10 and expressed as mean ± SD. Statistical differences between two groups were analyzed using two-tailed Student's t-tests. Statistical differences among three or more groups were analyzed using one-way ANOVA combined with the Brown-Forsythe test. A p-value less than 0.05 was considered statistically significant. 0.01 < P < 0.05 was indicated by *; 0.001 < P < 0.01 was indicated by **; P < 0.001 was indicated by ***; and P < 0.0001 was indicated by ****.

[0050] 2.2 Experimental Results 2.2.1 Effects of HP hydrogel scaffolds on RAW264.7 cell proliferation, antioxidant capacity, and polarization capacity The results showed that when the PUE concentration exceeded 0.6 wt%, the absorbance of CCK-8 cells decreased significantly with increasing PUE concentration, and cell viability decreased significantly. Figure 2 (A). To achieve a balance between biosafety and functional effects, this invention systematically screened different concentration gradients and ultimately determined the optimal PUE concentration to be 0.3 wt%. At this concentration, cell viability remained at 85%, and no significant cytotoxic reactions were observed.

[0051] ROS clearance assays showed that the Control group produced the least amount of intracellular ROS, representing the baseline oxidative stress level. The LPS model group showed significantly elevated ROS levels, the highest among the five groups, indicating that LPS successfully induced a significant oxidative stress response. In the intervention groups, the ROS levels in the HLS, PZn, and HP groups were all lower than those in the LPS model group, showing a decreasing trend. The HP group's ROS levels were close to those of the control group, suggesting that it had the most significant antagonistic effect against LPS-induced oxidative stress. Figure 2 (B). In addition, RT-qPCR ( Figure 2 (C) and immunofluorescence staining analysis ( Figure 2The results of the de novo cytometry (DE) studies showed that the HP group exhibited the most significant immune regulation. The HP scaffold effectively induced macrophage polarization towards the M2 morphology, increased the expression of Arg-1 and YM-1, and decreased the expression of iNOS and IL-1β. The iNOS expression level of the HP scaffold was 30% of that in the LPS group, and the Arg-1 expression level was 14 times that in the LPS group, indicating that it has a strong ability to promote the optimization of the bone repair microenvironment.

[0052] It is evident that the HP scaffold of the present invention exhibits strong antioxidant and anti-inflammatory capabilities, which may be closely related to the immunomodulatory role played by PUE in macrophages.

[0053] 2.2.2 HP hydrogel scaffold's ability to recruit bone marrow mesenchymal stem cells and promote osteogenic differentiation In addition to alleviating the inflammatory microenvironment of macrophages, CM treated with HP scaffolds also significantly promoted osteogenic differentiation of BMSCs. ALP and ARS staining results showed that on days 7 and 14 of culture on HP scaffolds, the ALP activity and calcium deposition in the HP group were significantly greater than those in the HLS and PZn groups. Figure 3 (AC). Immunofluorescence staining showed that the HP scaffold significantly promoted the expression of Col-1α and OCN, further demonstrating its role in promoting osteogenic differentiation. Figure 3 (DE). From the cytoskeleton and cell spreading state on the material surface, it can be seen that HP has the strongest ability to recruit BMSCs, with the most BMSCs adhering to its surface, and all exhibiting a flat spreading state, indicating good activity. Figure 3 (FG).

[0054] RT-qPCR results showed that the HP scaffold induced the upregulation of osteogenic marker genes such as Runx2, ALP, and BMP2, suggesting that it promotes bone formation by regulating related signaling pathways of osteogenic differentiation. Figure 3 (H). WB results were consistent with immunofluorescence staining results: compared with the Control group, HP showed a superior ability to promote the production of OCN, OPN, and Col-1α. Figure 3 Middle I).

[0055] The HP scaffold of this invention, through its unique spatial septation design, enables "immune-led" sequential osteogenic formation, and its core layer releases PUE and Zn. 2+ It provides a favorable immune microenvironment for osteogenic formation, and the sheath releases SDF-1α and Mg 2+ Improving the homing efficiency and osteogenic differentiation capacity of BMSCs to bone defect areas can significantly enhance bone regeneration without the need for additional implantation of a large number of seed cells.

[0056] 2.2.3 HP hydrogel scaffold's ability to promote vascularization Angiogenesis is a crucial step in osteogenic processes. Blood vessel formation not only provides essential nutrients and oxygen to bone tissue but also creates favorable conditions for osteoblast migration and new bone formation. Scratch-healing, tube formation, and migration assays showed that the HP scaffold significantly promoted the angiogenesis of HUVECs. The scratch-healing assay demonstrated that the HP scaffold significantly improved the horizontal migration ability of HUVECs, with a migration rate significantly higher than that of Control, HLS, and PZn within 6 hours. Figure 4 (A). Tube formation experiments showed that the HP scaffold promoted the formation of tubular structures of HUVECs on Matrigel. Figure 4 (B). Migration experiments showed that, compared to HLS and PZn, HP was more effective at inducing the vertical migration of HUVECs. Figure 4 (C). The statistical data (migration rate, number of vascular nodules, number of vascular branches, total vascular length, and number of migrating cells) showed the following trend across the three materials: HP > PZn > HLS. Figure 4 (D). Immunofluorescence staining results showed that the HP scaffold significantly increased CD31 expression, indicating that it promoted the vascularization process. Figure 4 (EF). The above analysis results show that the HP group has a significantly better advantage in vascularization than the PZn, HLS and Control groups, especially in the stability of tubular structures, the number of tubular segments and the rate of angiogenesis.

[0057] 2.2.4 Repair effect of HP hydrogel scaffold in osteoporosis rat model This invention further evaluates the effect of the HP scaffold on repairing osteoporosis in a rat model of osteoporosis. The results show that in the rat model of osteoporosis, the HP scaffold significantly promotes new bone formation at the site of bone defects. Significant differences in osteogenic effects were observed among the four groups, with the HP group showing superior osteogenic effects compared to the other groups. Micro-CT and three-dimensional reconstruction analyses showed that the HP group exhibited significantly more trabecular bone formation and denser bone tissue. Figure 5 (A). Further analysis of bone mineral density (BMD), bone volume / volume ratio (BV / TV), and trabecular bone number (Tb.N) showed ( Figure 5In the control group (B), the HP group showed significantly higher values ​​than the PZn, HLS, and Control groups in all these indicators, while the trabecular separation (Tb.Sp) of the HP group was lower than that of the other groups. Compared with the Control group, the HP group showed increases of 41%, 63%, and 46% in BMD, BV / TV, and Tb.N, respectively, while Tb.Sp decreased by half. These data indicate that the HP group not only has more bone tissue formation but also higher trabecular connectivity and bone density. HE staining and Masson staining results were also consistent with the above analysis, indicating that the HP group has a stronger bone formation capacity (B). Figure 5 (C) The heart, liver, spleen, lungs, and kidneys in all four groups showed intact structures with no obvious damage or inflammatory response. Figure 5 The results (D) indicate that the materials in each group showed good biocompatibility and safety in these organs.

[0058] Immunofluorescence staining results showed that the HP group had CD31 ( Figure 6 (A) and OCN ( Figure 6 The expression of Col-1α in the HP group was significantly higher than in other groups, indicating that it can effectively support the nutritional and oxygen supply to the bone repair area, thereby accelerating bone regeneration. Meanwhile, the high expression of Col-1α in immunohistochemical staining results showed that the HP group significantly promoted bone matrix synthesis and enhanced the stability and strength of bone structure. Figure 6 (D). In contrast, the PZn and HLS groups showed lower expression of these three markers, while the Control group showed almost no expression of angiogenesis and osteogenic markers, further validating the advantage of the HP group in promoting bone repair and vascularization. The HP group significantly promoted macrophage polarization to the M2 phenotype, manifested by high expression of the M2 marker Arg-1, while the expression of the M1 marker iNOS was significantly reduced (D). Figure 6 (C). In comparison, the PZn and HLS groups showed slightly weaker effects on M2 polarization, while the Control group showed almost no M2 polarization. This phenomenon indicates that the HP group promoted the repair and regulation of the immune microenvironment by regulating the polarization state of macrophages, thereby promoting bone repair and regeneration.

[0059] In the osteoporosis rat model of this invention, the HP group exhibited the most significant bone repair effect, which is likely attributed to the bone immune regulation effect mediated by puerarin. M2 macrophages can secrete anti-inflammatory cytokines, enhance endothelial cell migration and angiogenesis, and promote the differentiation of mesenchymal stem cells towards osteogenic processes, thereby effectively promoting angiogenesis and bone formation. PUE, by inhibiting pro-inflammatory signals such as NF-κB / MAPK, reducing oxidative stress, and enhancing the anti-inflammatory environment, helps to construct an immune microenvironment that promotes angiogenesis and bone regeneration.

[0060] 2.2.5 HP hydrogel scaffolds promote osteoporotic bone defect repair by remodeling arginine metabolism in RAW 264.7 cells and activating the mTOR pathway in BMSCs. To elucidate the mechanism by which the HP hydrogel scaffold promotes osteoporosis repair, this invention performed transcriptome sequencing analysis on different treatment groups. PCA analysis showed that the HP group and the Control group were significantly separated at the overall transcriptome level. Figure 7 (A) This suggests that the HP scaffold significantly remodeled the gene expression patterns of local cells. The volcano plot results showed a large number of differentially expressed genes in the HP group (…). Figure 7 In group B, 493 genes were upregulated and 761 genes were downregulated. The heatmap further validated that the differentially expressed genes exhibited consistent and distinguishable expression clustering characteristics between the two groups. Figure 7 (C) The genes of amino acid transporter SLC7A14 and lysosomal-associated proteins ATP6V0A1 and p62 showed a significant upregulation trend in the HP group. The expression levels of SLC7A14, ATP6V0A1, and p62 genes in the HP group were 5.5, 2.1, and 2.1 times higher than those in the Control group, respectively. Figure 7 (Figure 7E-F). GO enrichment analysis showed that differentially expressed genes were mainly enriched in metabolic regulation and biosynthesis-related processes, including ATP-dependent activity, chromosomal region, and DNA replication (Figure 7E-F). KEGG enrichment analysis further indicated that the HP group was significantly enriched in pathways such as DNA replication, amino acid metabolism, and the Immune system, and also involved replication and repair-related networks (Figure 7E-F). Figure 7 GH). GSEA enrichment analysis showed a positive enrichment trend in the Positive Regulation of DNA Replication and Cellular Amino Acid Biosynthesis Process pathways. Figure 7 (I) This further illustrates that the HP scaffold induces a transcriptional activation state characterized by DNA replication and amino acid synthesis. HP treatment of RAW264.7 cells upregulated the expression of lysosome-related proteins ATP6V0A1 and SLC7A14, suggesting enhanced lysosomal acidification, amino acid transport, and reabsorption, indicating that HP can promote lysosomal functional remodeling. Simultaneously, p62 increased, and further accumulated after BafA1 treatment; LC3-I decreased while LC3-II increased, with greater changes after BafA1 treatment, indicating that HP promotes autophagosome formation and autophagy flux, rather than simply inhibiting degradation. Figure 7 (Middle K).

[0061] The transcriptomic results above suggest that, compared with simple bone mass enhancement, the HP scaffold also exhibits a systemic remodeling of immune and metabolic-related molecular pathways. The HP group significantly enhanced energy metabolism, amino acid metabolism, and nucleotide metabolism pathways, indicating that cells are more inclined to enhance anabolic metabolism and be in a functionally activated state. This characteristic is consistent with the repair phenotype observed in histological and immunological markers. Enhanced amino acid and nucleotide metabolism helps support the proliferation, secretion, and matrix synthesis needs of cells during the repair period, thereby synergistically promoting angiogenesis and osteogenic processes. Particularly noteworthy is that puerarin, by enhancing lysosomal function and autophagic flux, promotes the recycling of degradation products, especially amino acids, providing metabolic support for maintaining cellular homeostasis. Therefore, the superior repair effect exhibited by the HP scaffold in the osteoporotic microenvironment may stem from its mediated co-activation of immunometabolic regulation and amino acid transport pathways.

[0062] Metabolomics analysis further revealed the metabolic basis of the HP hydrogel scaffold's role in promoting bone repair under osteoporotic conditions. PCA analysis showed that ( Figure 8 (A) The HP group and the Control group showed a clear separation in the overall metabolic profile, suggesting that the HP scaffold significantly remodeled the local metabolic state. (Volcano plot) Figure 8 (B) and bubble scatter plot ( Figure 8 The results (C) indicated that multiple differentially metabolites, including organic acids and derivatives, were significantly upregulated in the HP group. GO and KEGG enrichment analyses further showed that these differentially metabolites were mainly concentrated in pathways related to amino acid metabolism and synthesis. Figure 8 (DE), suggesting that the HP scaffold participates in bone regeneration regulation by activating an amino acid metabolic network. Heatmap analysis ( Figure 8 The results from the study showed that the three arginine metabolites, Arginine, Ornithine, and Putrescine, were significantly enriched in the HP group, and quantitative analysis showed that ( Figure 8 In the M2 group, the levels of arginine, ornithine, and putrescine were 2.0, 3.4, and 1.9 times higher than in the control group, respectively, indicating that arginine metabolism may play a key role in HP-mediated osteogenic processes. The key arginine metabolism enzymes Arg-1 and ODC-1 were upregulated in the HP group, while iNOS was downregulated, a trend consistent with the M2 group. Figure 8 (H).

[0063] To further verify the function of arginine metabolism in the osteogenic process of hematopoietic stem cells (HP), this invention used macrophage supernatant supplemented with L-Arg and nor-NOHA to functionally intervene in BMSCs, followed by RT-qPCR ( Figure 8 I) and WB Figure 8The expression of osteogenic markers and mTOR pathway-related signals were analyzed and detected. Results showed that the expression levels of OPN and Col-1α in the HP group were significantly higher than those in the Control group, HP+nor-NOHA group, and HP+nor-NOHA+L-Arg group. After the addition of nor-NOHA, the expression of osteogenic-related genes and proteins significantly decreased; however, supplementation with exogenous arginine partially restored their expression levels. HP upregulated the expression levels of mTOR pathway-related proteins p-mTOR and p-S6, while the expression of these proteins was significantly inhibited by nor-NOHA. Further addition of L-Arg to the system increased the expression levels of p-mTOR and p-S6. These results fully demonstrate that arginine undergoes metabolic diversion, preferentially entering the polyamine synthesis pathway through the upregulation of Arg-1 and ODC to promote putrescine secretion. Putrescine acts as a key metabolic paracrine signaling agent, enhancing the synthesis of osteogenic differentiation-related proteins in BMSCs and activating the mTOR signaling pathway. Ultimately, it promotes the remodeling of the bone regeneration immune microenvironment and accelerates bone repair, providing sufficient metabolic substrates and signaling support for osteogenic differentiation.

[0064] The pro-repair effect of HP hydrogel in the osteoporotic environment may essentially be a microenvironment regulation process centered on immune metabolic reprogramming, rather than simply providing bone defect filling or osteogenic support. In summary, HP is more likely to act first. Enhancing the lysosome-autophagy axis improves the cell's ability to degrade, recycle, and redistribute endogenous substrates, thereby continuously providing metabolic substrates such as arginine to the local microenvironment. Furthermore, arginine metabolism shifts towards Arg-1 / ODC-mediated polyamine synthesis rather than the iNOS-dominated inflammatory metabolic pathway. This reflects not only the functional shift of macrophages from a pro-inflammatory to a repair phenotype but also suggests that metabolic shunting itself may be a crucial link between immune regulation and bone regeneration. Moreover, putrescine is not merely a end product of arginine metabolism but also acts as a paracrine metabolic signal, participating in the regulation of protein synthesis and osteogenic processes in bone mesenchymal stem cells (BMSCs), with the mTOR pathway being a key downstream node. Thus, the bone repair process mediated by HP hydrogel through immune microenvironment reconstruction exhibits a transcellular metabolic coupling mechanism: enhanced macrophage metabolism leads to arginine shunting to the polyamine pathway, thereby activating osteogenic signaling in BMSCs.

[0065] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. An immune-metabolic regulation unzipping gel scaffold, characterized in that, The support has a core-sheath structure; The core layer of the core-sheath structure comprises methacrylamide hyaluronic acid, lithium magnesium silicate, stromal cell-derived factor-1α, and a photoinitiator. The sheath layer of the core-sheath structure contains puerarin and zinc ions.

2. The immuno-metabolic regulation unzipping gel scaffold of claim 1, wherein, The photoinitiator is I2959.

3. A method for preparing the immune-metabolic regulation unblocking gel scaffold according to claim 1 or 2, characterized in that, Includes the following steps: Step 1: Methacrylamide hyaluronic acid, lithium magnesium silicate, matrix cell-derived factor-1α and photoinitiator are dissolved in deionized water to prepare the core layer bio-ink. Step 2: Disperse puerarin in deionized water, heat to dissolve, cool down, add zinc salt, stir evenly, and prepare sheath bio-ink. Step 3: Load the core layer bio-ink from Step 1 and the sheath layer bio-ink from Step 2 into the inner and outer cylinders of the coaxial 3D printer, respectively, and print using the coaxial nozzle. Step four: The scaffold printed in step three is subjected to photocuring and cross-linking to obtain a gel scaffold with a core-sheath structure.

4. The preparation method according to claim 3, characterized in that, The concentration of the methylacrylated hyaluronic acid in the core bio-ink is 1-4 wt%, the concentration of the magnesium lithium silicate is 2-8 wt%, the concentration of the stromal cell-derived factor-1α is 1×10 -5 -1×10 -4 wt%, and the concentration of the photoinitiator is 0.1-0.5 wt%. The concentration of the puerarin in the sheath bio-ink is 1-10 wt%, and the concentration of the zinc salt is 0.5-2 wt%.

5. The preparation method according to claim 4, characterized in that, The concentration of the methylacrylated hyaluronic acid in the core bio-ink is 4 wt%, the concentration of the magnesium lithium silicate is 4 wt%, the concentration of the stromal cell-derived factor-1 alpha is 5×10 - 5 wt%, and the concentration of the photoinitiator is 0.2 wt%; the concentration of the puerarin in the sheath bio-ink is 4 wt%, and the zinc salt is ZnSO4·7H2O and the concentration is 1 wt%.

6. The preparation method according to claim 3, characterized in that, In step two, the heating and dissolution conditions are: heating at 95–100 °C for 5–15 minutes; adding zinc salt when the temperature drops to 30–40 °C.

7. The preparation method according to claim 3, characterized in that, In step three, the printing speed is 400–700 mm / s, and the platform temperature is -10–0 °C; the mixing ratio of the core layer bio-ink to the sheath layer bio-ink is 6:

4.

8. The preparation method according to claim 3, characterized in that, In step four, the wavelength of the light source for photocuring crosslinking is 405 nm, and the irradiation time is 0.5–3 min.

9. The use of the immunomodulatory desealing gel scaffold as described in claim 1 or 2 in the preparation of products for the repair of osteoporotic bone defects.

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