Intelligent biological battery for diabetic bone defect regeneration as well as preparation method and application of intelligent biological battery

Abstract

The invention belongs to the field of diabetic bone defect regeneration, and particularly relates to an intelligent biological battery for diabetic bone defect regeneration and a preparation method and application thereof. The preparation method comprises the following steps: respectively assembling a tetrafluorophenylboronic acid-loaded methacrylated gelatin hydrogel scaffold and an osteostatin-loaded methacrylated gelatin hydrogel scaffold, and tightly integrating a conductive graphene oxide electrostatic spinning membrane and the hydrogel scaffold through a self-assembly process to obtain the hydrogel scaffold. And the intelligent biological battery for diabetic bone defect regeneration is obtained. The intelligent biological battery disclosed by the invention can spontaneously generate and adjust the magnitude of micro current according to blood sugar fluctuation, adjust an immune microenvironment and an osteogenesis-osteoclast axis, promote angiogenesis and clear ROS, and can be used for bone regeneration treatment of diabetic patients.

Description

Technical Field

[0001] The present invention belongs to the field of diabetic bone defect regeneration, and particularly relates to an intelligent biobattery for diabetic bone defect regeneration, a preparation method thereof, and an application thereof. Background Art

[0002] Diabetes mellitus (DM) is a metabolic disorder disease characterized by hyperglycemia and chronic inflammation. A large number of studies have shown that due to the inhibition of osteogenic differentiation, the deterioration of the bone tissue microenvironment, and abnormal inflammatory responses, the treatment of critical-sized bone defects caused by tumor resection or trauma in diabetic patients is more challenging than that in healthy individuals. In the diabetic microenvironment, the abnormal elevation of pro-inflammatory cytokines such as interleukin (IL)-1 and IL-6, as well as the imbalance of M1 / M2 macrophage polarization, are the key factors leading to cell apoptosis and the inhibition of osteoblast differentiation. Traditional bone repair methods for patients with normal blood glucose often have poor effects in diabetic patients. With the rapid growth of the diabetic population, the demand for effective bone repair strategies is increasing day by day, highlighting the urgency of developing efficient methods to accelerate bone regeneration in diabetic patients.

[0003] Currently, it is generally recognized in the academic community that immune disorders significantly exacerbate the delayed healing of diabetic bone injuries. Diabetic hyperglycemia can induce immune cell dysfunction, and macrophages play a key role in regulating inflammation and bone tissue healing. Macrophages in the bone microenvironment are roughly divided into M1 and M2 phenotypes. At the initial stage of bone defect healing, M1 macrophages dominate, responsible for clearing cell debris and secreting chemotactic signals such as monocyte chemoattractant protein-1 (MCP-1) and C-X-C motif chemokine ligand 8 (CXCL8). These factors play a key role in recruiting bone marrow mesenchymal stem cells (BMSCs), bone progenitor cells, and vascular progenitor cells to the injury site. As the healing process enters the tissue repair stage, the number of M2 macrophages gradually increases, releasing growth factors including interleukin-10 (IL-10), bone morphogenetic protein-2 (BMP-2), and arginase-1, which jointly promote bone regeneration. However, in the diabetic pathological microenvironment, the acute inflammation period is significantly prolonged, accompanied by the continuous activation of M1 macrophages, resulting in insufficient conversion of M1 to M2 phenotype, causing a serious imbalance in the ratio of M1 / M2 macrophages and hindering tissue repair. In addition, the pro-inflammatory cytokines secreted by M1 macrophages induce apoptosis of bone marrow mesenchymal stem cells and osteoblasts through regulating multiple signaling pathways and promote osteoclastogenesis, thereby inhibiting osteogenic differentiation. The weakened repair function of M2 macrophages ultimately leads to bone repair failure.

[0004] In addition to immune disorders, the pathological features of diabetes also include mitochondrial dysfunction, abnormal activation of energy metabolism, and impairment of the intracellular antioxidant defense system, ultimately leading to excessive production of reactive oxygen species (ROS), which severely impairs bone injury healing in diabetic patients. ROS can interact with macrophages. Excessive ROS not only directly inhibits osteogenic differentiation through the PI3K / AKT signaling pathway but also forms a mutually promoting relationship with the continuous polarization of M1 macrophages, constituting a vicious cycle that ultimately damages bone formation. Therefore, regulating the immune microenvironment in bone tissue and protecting cells at the injury site from oxidative stress damage are crucial for successful bone repair.

[0005] Increasing studies have shown that physiological electrical signals play an indispensable role in maintaining the homeostasis of the bone microenvironment. Research has shown that electrical stimulation (ES) can regulate cell activity, enhance tissue repair, and promote angiogenesis and bone regeneration. In addition, ES has immunomodulatory effects and the ability to scavenge ROS. As a novel biophysical means, ES has a multi-faceted impact on tissue regeneration. Utilizing electroactive biomaterials to mimic the bioelectrical properties of healthy bone tissue - thus providing a favorable microenvironment for bone regeneration under diabetic conditions - represents a promising research direction. However, current research mainly focuses on the direct effects of ES on the proliferation and differentiation of osteoblasts, neglecting its potential immunomodulatory and ROS scavenging effects, especially in the pathological microenvironment of diabetes. In addition, traditional endogenous battery devices are usually bulky, have poor current stability, and are difficult to seamlessly integrate with the natural biological processes of the human body. Emerging piezoelectric materials such as barium titanate and lead zirconate titanate usually require continuous mechanical stress or frictional stimulation to generate electrical signals. However, early after fracture surgery, patients need local immobilization, and limited mechanical stress results in the inability to effectively activate the piezoelectric or triboelectric properties of these materials. This causes the crucial early immune regulation stage of bone defect healing to be missed, and the early immune microenvironment at the fracture site cannot be improved. Therefore, developing bone immune regulatory repair materials that can dynamically respond to fluctuations in blood glucose levels without external mechanical stimulation - with multifunctional properties such as immunomodulation, bone induction, and osteoclast inhibition - is a highly promising strategy.

[0006] Based on the above background, we developed an intelligent GF-Os G bio-battery system. The system is assembled through GelMA hydrogel scaffolds loaded with tetrafluorobenzeneboronic acid (FPBA) and osteostatin respectively, and then the conductive reduced graphene oxide electrospun membrane is tightly integrated with the hydrogel scaffold through a self-assembly process. When the GF-Os G system is implanted into the skull defect site of diabetic rats, the glucose-responsive tetrafluorobenzeneboronic acid maintains a stable negative charge state, while osteostatin enhances its positive charge state. This process establishes an endogenous electric field, and subsequent charge migration generates a microcurrent, ultimately promoting angiogenesis, scavenging ROS, regulating macrophage polarization, and accelerating bone defect healing. Summary of the Invention

[0007] To solve the problems existing in the intelligent biological battery system in the prior art, the present invention provides an intelligent GF-Os G biological battery system. The system is assembled by GelMA hydrogel scaffolds loaded with tetrafluorobenzeneboronic acid (FPBA) and osteostatin respectively, and then the conductive graphene oxide electrospun membrane is tightly integrated with the hydrogel scaffold through a self-assembly process. When the GF-Os G system is implanted into the skull defect site of diabetic rats, the glucose-responsive tetrafluorobenzeneboronic acid maintains a stable negative charge state, while osteostatin strengthens its positive charge state. This process establishes an endogenous electric field, and subsequent charge migration generates a microcurrent, which ultimately promotes angiogenesis, scavenges ROS, regulates macrophage polarization, and accelerates bone defect healing. The intelligent GF-Os G biological battery system can spontaneously generate and regulate the magnitude of the microcurrent according to blood glucose fluctuations, regulate the immune microenvironment and the osteoblast-osteoclast axis, and promote angiogenesis and scavenge ROS. It is beneficial to the effective design of functionalized biomaterials for the bone regeneration treatment of diabetic patients.

[0008] The technical solution provided by the present invention is:

[0009] A preparation method of an intelligent biological battery for diabetic bone defect regeneration, the preparation method comprising: assembling a methacrylated gelatin hydrogel scaffold loaded with tetrafluorobenzeneboronic acid and a methacrylated gelatin hydrogel scaffold loaded with osteostatin respectively, and then tightly integrating the conductive graphene oxide electrospun membrane with the hydrogel scaffold through a self-assembly process to obtain an intelligent biological battery for diabetic bone defect regeneration.

[0010] Further, the self-assembly process comprises: placing the methacrylated gelatin hydrogel scaffold loaded with tetrafluorobenzeneboronic acid and the methacrylated gelatin hydrogel scaffold loaded with osteostatin on both sides of a separator membrane; laminating the conductive graphene oxide electrospun membrane onto the surfaces of the methacrylated gelatin hydrogel scaffold loaded with tetrafluorobenzeneboronic acid and the methacrylated gelatin hydrogel scaffold loaded with osteostatin, and assembling them tightly after close fitting to obtain an intelligent biological battery for diabetic bone defect regeneration.

[0011] Further, the preparation method of the conductive graphene oxide electrospun membrane comprises: slowly adding polyvinyl butyral to absolute ethanol, fully dissolving it, adding a graphene oxide dispersion liquid, and fully stirring and mixing to obtain an electrospinning solution, and preparing a conductive graphene oxide electrospun membrane.

[0012] Further, the preparation method of the graphene oxide dispersion liquid comprises: adding graphene oxide powder to deionized water, fully mixing, and performing ultrasonic treatment under ice-water bath conditions to obtain a uniformly dispersed graphene oxide solution.

[0013] Furthermore, the preparation method of the tetrafluorophenylboric acid-loaded methacrylylated gelatin hydrogel scaffold includes: dissolving freeze-dried methacrylylated gelatin and tetrafluorophenylboric acid in a dimethyl sulfoxide aqueous solution and fully mixing them, then adding a photoinitiator to the mixed solution, stirring until completely dissolved, and completing photocrosslinking after ultraviolet irradiation to obtain a tetrafluorophenylboric acid-loaded methacrylylated gelatin hydrogel scaffold.

[0014] Furthermore, the preparation method of the methacryloyl gelatin hydrogel scaffold loaded with osteostatin includes: dissolving freeze-dried methacryloyl gelatin and osteostatin in PBS and fully mixing, then adding a photoinitiator to the mixed solution, stirring until completely dissolved, and completing photocrosslinking after ultraviolet irradiation to obtain a methacryloyl gelatin hydrogel scaffold loaded with tetrafluorophenylboric acid.

[0015] The present invention also provides an intelligent bio-battery for diabetic bone defect regeneration, which is prepared by the above-mentioned preparation method.

[0016] The present invention also provides the use of the above-mentioned intelligent biobattery for diabetic bone defect regeneration in the preparation of bone immune regulation repair materials.

[0017] The present invention also provides the use of the above-mentioned intelligent biobattery for diabetic bone defect regeneration in reducing cellular ROS levels.

[0018] The present invention also provides the use of the above-mentioned intelligent biobattery for diabetic bone defect regeneration in improving the ratio of M2 / M1 macrophages.

[0019] The present invention also provides the regulation of immunity and osteoblast-osteoclast axis, as well as the construction and functional characterization verification of an angiogenesis complex system.

[0020] This invention is to solve the key problems that the chronic inflammatory microenvironment under the pathological environment of diabetes is not conducive to bone defect repair and the osteogenic microenvironment is dynamically adjusted under the blood sugar fluctuation environment. A hydrogel with glucose response is designed and prepared, which can adjust the surface charge according to the blood sugar concentration. And electrospinning of graphene oxide with good conductivity is used to gradually build a biomimetic microenvironment responsive smart biobattery. FPBA on one side of the hydrogel is expected to respond to the high glucose environment, causing the charge to change from neutral to negative. Osteostatin on the other side of the hydrogel can enhance the positive charge capacity in the weakly acidic environment of diabetic inflammation. As a result, a potential difference is formed on both sides, and a microcurrent is transmitted through the graphene oxide conductive spinning membrane, polarizing macrophages to the M2 phenotype, inducing the secretion of factors related to osteogenesis and angiogenesis, inhibiting osteoclasts, and clearing ROS. The smart biobattery has multiple functions, such as glucose response, bone immune regulation, bone induction, angiogenesis and ROS clearance.

[0021] In a first aspect, the present invention constructs an intelligent biobattery for dynamically regulating the immune microenvironment in a diabetic environment, and utilizes the charge changes of FPBA and Osteostatin in the diabetic inflammatory microenvironment to generate a microcurrent ( Figure 1 a).

[0022] In a second aspect, the present invention verifies the ability of the composite material system to regulate immunity and osteogenesis and angiogenesis, as well as the therapeutic effect of the system in a bone defect model of diabetic rats ( Figure 1 b).

[0023] The pathological diabetic microenvironment consists of glucose fluctuations and chronic inflammation, resulting in delayed healing of bone defects. Therefore, it is necessary to develop new materials that are beneficial to bone integration and suitable for use in the complex diabetic microenvironment. This study reports a novel glucose-responsive GF-Os G intelligent biobattery. This composite can regulate the local immune microenvironment and coordinate the osteoblast-osteoclast axis, promote angiogenesis and scavenge ROS. The study found that GF-Os G promotes osteogenesis through the ERK / P38-GPX4 axis, effectively scavenges excess free radicals, and promotes osteogenic differentiation. Overall, the findings obtained indicate that GF-Os G regulates the immune microenvironment and enhances the repair effect of bone defects in diabetic patients. In addition, the present invention provides an effective strategy for designing functional biomaterials for the treatment of bone regeneration in diabetic patients. Brief Description of the Drawings

[0024] Figure 1 Schematic diagram for GF-Os G to promote diabetic bone regeneration, where a is the preparation schematic diagram of GF-Os G ; b is the mechanism diagram of GF-Os G to promote the repair of diabetic bone defects;

[0025] Figure 2 Physical and chemical characterization of the GF-Os G biobattery, where a is the preparation schematic diagram of GF-Os G . b is the SEM photos of the surface morphology of G, GF, G-Os and Go 3 ; c is the EDS elemental map of GF; d is the Fourier transform infrared spectrum (FTIR); e is the XPS spectrum of GF-Os G ; f is the degradation rate of the GF-Os G biobattery in a high glucose environment (37 °C; pH 7.4; high glucose: 25.0 mmol L-1; low glucose: 5.6 mmol L-1); g is the drug release curve (37 °C; pH 7.4; high glucose: 25.0 mmol L-1 ; Low glucose: 5.6 mmol / L -1 ), h is the mechanical strength curve, and i is the elastic modulus and storage modulus of the scaffold;

[0026] Figure 3 is GF-Os G The electrical characteristics of the bio-battery, where a is the schematic diagram of microcurrent generation, b is the cyclic voltammetry characteristic curve of Go 3 of, c is Go 3 and Go 2 of the electrochemically active area analysis, d is Go 3 and Go 2 of the linear voltammetry characteristic curve composite diagram, e is Go 3 and Go 2 of the general overpotential diagram, f is Go 3 and Go 2 of the Tafel curve, g is Go 3 and Go 2 of the Nyquist diagram, h is Go 3 and Go 2 of the conductivity, i is the microcurrent detected after 1 week of degradation of GF-OsG in a high glucose environment;

[0027] Figure 4 is the regulation of the polarization phenotype of macrophages in vitro, where a is BMDMs stained with CD86 (red), CD206 (green) and DAPI (blue), b is the flow cytometry analysis of CD86 and CD206 intracellular in BMDMs, c-f are further detection of the expression of pro-inflammatory genes (TNF-α, iNOS) and anti-inflammatory genes (IL-10, TGF-β) by quantitative RT-qPCR analysis, g-h are representative images of WB and quantitative analysis of CD86, iNOS, CD206 and ARG1, i is the ELISA analysis of TNF-α, IL-6, TGF-β and IL-10 levels;

[0028] Figure 5Effects of various scaffolds on in vitro osteogenesis and osteoclastogenesis. Among them, a shows the ALP staining results of BMSCs co-cultured with different scaffolds for 7 days, b shows the ALP activity of BMSCs co-cultured with different scaffolds for 7 days, c-d show the ARS staining and quantitative analysis of calcium deposition of BMSCs co-cultured with different scaffolds for 21 days, e shows the expression of osteogenesis-related genes of BMSCs co-cultured with different scaffolds for 7 days, e shows the TRAP staining map of BMDMs co-cultured with different scaffolds under calcification conditions, e shows the expression of bone formation-related genes in BMSCs co-cultured with different scaffolds for 7 days, f shows the TRAP staining images of BMDMs co-cultured with different scaffolds in the presence of M-CSF (30 ng / mL) and RANKL (50 ng / mL), g shows the number of TRAP-positive osteoclasts, h shows the SEM images of the bone resorption area of BMDMs co-cultured with different scaffolds, i shows the relative bone resorption area analyzed quantitatively by Image J, j shows the representative immunofluorescence staining images of F-actin of osteoclasts co-cultured with different scaffolds, k shows the quantification of actin ring (+) osteoclasts by Image J, and l shows the quantitative analysis of the expression of osteoclast differentiation genes;

[0029] Figure 6 is GF-Os G In vivo regulation of bone remodeling by bio-battery. Among them, a is a schematic diagram, b are representative three-dimensional reconstructed micro-CT images of rat skull defects at 4 weeks and 8 weeks after surgery, c is the quantitative analysis of new bone formation based on micro-CT parameters 4 weeks and 8 weeks after implanting the scaffold, and d are H&E and Masson's trichrome staining of new bone 4 weeks and 8 weeks after implanting the scaffold;

[0030] Figure 7 Immunohistochemical analysis of diabetic rats. Among them, a are immunohistochemical stains of TNF-α and TGF-β in the defect area, b is the quantitative analysis of TNF-α and TGF-β positive areas by Image J, c are immunohistochemical stains of VEGF and CD31 in the defect area, d-e are the quantitative analysis of VEGF positive (d) and CD31 positive (e) areas by Image J, f are immunohistochemical stains of COL1 and RUNX2 in the defect area, g-h are the quantitative analysis of COL1 positive (g) and RUNX2 positive (h) areas by Image J, and i is the immunohistochemical stain of TRAP in the defect area. J is the quantitative analysis of the TRAP positive area by Image J;

[0031] Figure 8 Transcriptome sequencing and western blot experimental study of GF-Os G Mechanism of bio-battery regulating osteogenic differentiation. Among them, a is between Ctrl and GF-OS G Pearson correlation matrix, b is between Ctrl and GF-OsG Volcano plot of differentially expressed genes between them. c shows the enriched signaling pathways based on significantly changed genes in BMSCs. d shows the top 20 KEGG enrichments related to differentially expressed genes by KEGG enrichment analysis. e is the GO enrichment analysis bubble plot. f shows the top 20 enriched biological processes related to differentially expressed genes by GO enrichment analysis. g is GF-Os G Schematic diagram of the mechanism promoting osteogenesis and alleviating intracellular reactive oxygen species (ROS) levels; h-k are the results of Western blot;

[0032] Figure 9 Total photos of GF-Os and GF-OsG;

[0033] Figure 10 EDS elemental mapping of GelMA (G);

[0034] Figure 11 EDS elemental mapping of GF;

[0035] Figure 12 EDS elemental mapping of G-Os;

[0036] Figure 13 EDS elemental mapping of Go3;

[0037] Figure 14 XPS analysis of G, GF, G-Os and GF-Os;

[0038] Figure 15 Supplementary physical characterizations, where a is the swelling rate and b is the viscosity property of the scaffold;

[0039] Figure 16 Cyclic voltammetry characteristic curve of Go2;

[0040] Figure 17 Material current stability test, where a-b are Go 3 and Go 2 Composite graph of LSV curves at 1000 cycles and 1 cycle;

[0041] Figure 18 Photocurrent stability test, where a-b are Go 3 and Go 2 Composite graph of photocurrent stability test within 2400 seconds;

[0042] Figure 19 Zeta potential test of GF and G-Os hydrogels measured under different glucose conditions, where a-c are the Zeta potentials under different conditions and d is the histogram of zeta potential;

[0043] Figure 20 For microcurrent detection tests, where a - c are the microcurrents detected from GF - OsG after 2, 3, and 4 weeks of degradation in a high - glucose environment respectively;

[0044] Figure 21 It is the current detection graph of GF - OsG in a normal environment;

[0045] Figure 22 For the biocompatibility test of BMSCs, where a shows the live cells (green) and dead cells (red) of BMSCs co - cultured with different scaffolds, and b shows the quantitative analysis of the cell viability of BMSCs based on live / dead staining (n = 5 for each group, data are provided as standard deviation [SD] ± mean, *P < 0.05, **P < 0.01, ***P < 0.001);

[0046] Figure 23 To evaluate the cell viability of BMSCs co - cultured with different scaffolds by the CCK - 8 method; (n = 5 for each group, data are provided as standard deviation [SD] ± mean, *P < 0.05, **P < 0.01, ***P < 0.001);

[0047] Figure 24 For the ROS test, where a shows the representative fluorescence images of cellular ROS after co - culture with different scaffolds: green (ROS) and blue (DAPI), and b shows the fluorescence intensity of ROS evaluated by Image J (n = 5 for each group, data are provided as standard deviation [SD] ± mean, *P < 0.05, **P < 0.01, ***P < 0.001);

[0048] Figure 25 For the flow cytometry analysis of the intracellular ROS level in BMSCs co - cultured with different scaffolds;

[0049] Figure 26 For the biocompatibility test of BMDM, where a shows the live cells (green) and dead cells (red) of BMDM co - cultured with the scaffold, and b shows the quantitative analysis of the cell viability of BMDM based on Live / Dead staining (n = 5 for each group, data are provided as standard deviation [SD] ± mean, *P < 0.05, **P < 0.01, ***P < 0.001);

[0050] Figure 27 For the cell migration and angiogenesis assay, where a shows the crystal violet staining of HUVECs that migrated to the lower chamber of the transwell plate after 12 - hour culture, and b shows the quantitative analysis of the migrated cells. (n = 5 for each group, data are provided as standard deviation [SD] ± mean, *P < 0.05, **P < 0.01, ***P < 0.001);

[0051] Figure 28For the tube formation experiment, a is the tube formation assay of HUVEC co-cultured with the scaffold, and b is the quantitative analysis of tube length by Image J (n = 5 for each group, data are provided as mean ± standard deviation [SD], *P < 0.05, **P < 0.01, ***P < 0.001);

[0052] Figure 29 For the immunofluorescence staining experiment of HUVEC, where a is the fluorescence staining: green (CD31) and blue (DAPI), and b is the evaluation of the fluorescence intensity of CD31 by Image J (n = 5 for each group, data are provided as mean ± standard deviation [SD], *P < 0.05, **P < 0.01, ***P < 0.001);

[0053] Figure 30 For the immunofluorescence of BMSCs, where a is the immunofluorescence staining of BMSCs cells cultured for 3 days: green (COL1), red (phalloidin), and blue (DAPI), and b is the evaluation of the fluorescence intensity of COL1 by Image J (n = 5 for each group, data are provided as mean ± standard deviation [SD], *P < 0.05, **P < 0.01, ***P < 0.001);

[0054] Figure 31 For the experiment of BMSCs co-cultured with different scaffolds for 7 days, where a - c are the representative images of WB (a) and the quantitative analysis of COL1A1 (b) and RUNX2 (c) respectively (n = 5 for each group, data are provided as mean ± standard deviation [SD], *P < 0.05, **P < 0.01, ***P < 0.001);

[0055] Figure 32 For the network pharmacology model of Osteostatin, where a is the Venn diagram, b is the PPI network analysis of Osteostatin, and c - d are the GO enrichment analysis and KEGG pathway enrichment analysis respectively;

[0056] Figure 33 For the molecular semi-flexible docking of Osteostatin with HDAC4, SIRT1, KAT2B, and EP300;

[0057] Figure 34 For the experiment of BMDMs co-cultured with different scaffolds for 7 days in the presence of M-CSF (30 ng / mL) and RANKL (50 ng / mL), where a - c are the representative images of WB (a) and the quantitative analysis of MMP9 (b) and NFATC1 (c) respectively. (n = 5 for each group, data are provided as mean ± standard deviation [SD], *P < 0.05, **P < 0.01, ***P < 0.001);

[0058] Figure 35 H&E staining of the lung, kidney, spleen, liver, and heart at 8 weeks after stent implantation; scale bar = 100 μm;

[0059] Figure 36 Sequencing map, where a is for Ctrl and GF-Os G Principal component analysis (PCA), and b is a heat map of genes with significant changes between Ctrl and GF-OsG;

[0060] Figure 37 Sequencing map, where a - b are GSEA maps of the "growth factor binding" (a) and "extracellular exosome" (b) pathways, respectively;

[0061] Figure 38 Sequencing map, where a is the top 30 enriched gene sets in the gene set enrichment analysis (GSEA) of BMSCs ranked by p-value, and b is the main pathways related to differentially expressed genes shown by KEGG pathway enrichment analysis. Detailed implementation mode

[0062] Example 1

[0063] GF-Os G Preparation

[0064] The synthesis steps of methacrylated gelatin (GelMA) are as follows: Dissolve 20 g of gelatin in 200 mL of PBS and continuously stir at 60 °C for 2 hours. Subsequently, slowly inject 1 mL of methyl methacrylate into the gelatin solution through an aqueous membrane, and repeat this process 16 times while continuously stirring for 2 hours to form GelMA. Then dilute the reaction mixture with 800 mL of preheated PBS and gently stir for 15 minutes. To remove the unreacted methyl methacrylate, transfer the solution to a dialysis bag (cut-off molecular weight: 10,000 Da) and dialyze with deionized water for 7 days, changing the water twice a day. After dialysis, freeze the solution at -80 °C for 48 hours and lyophilize to obtain dry GelMA. Collect the dried product and seal it in a 50 mL centrifuge tube for later use. Dissolve 50 mg of lyophilized GelMA and 5 mg of tetrafluorobenzeneboronic acid (FPBA, C6H6BFO2, purchased from Macklin, China) in 1 mL of deionized water containing 0.1% dimethyl sulfoxide (DMSO), and mix well. Add the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (1 wt-%, purchased from Sigma, USA) and stir until completely dissolved. Place a transparent plastic separator film in the center of the silicone mold as a physical barrier, and pour the GelMA / FPBA (GF) solution onto one side of the mold. This separator film is used to physically separate the two parts and prevent direct electron transfer, thereby ensuring that the microcurrent only passes through the conductive graphene oxide electrospun membrane prepared by the following method, avoiding short circuits.

[0065] Subsequently, 50 mg of freeze-dried GelMA and 5 mg of osteostatin were dissolved in 1 mL of PBS and mixed thoroughly. The same concentration of photoinitiator (1 wt-%) was added. The GelMA / osteostatin (G-Os) solution was poured into the other side of the mold. Both sides were irradiated with UV light for 30 seconds to complete the photocrosslinking.

[0066] Weigh 1g of graphene oxide powder (using a precision electronic balance) and slowly add it to a beaker containing 100mL of deionized water to avoid direct agglomeration of the powder. Stir with a magnetic stirrer for 30 minutes during the addition process for preliminary mixing. Place the preliminarily stirred mixture in an ultrasonic cleaner and ultrasonicate at 300W for 30 minutes. Use an ice water bath to cool down during the ultrasonic process. After ultrasound, a uniformly dispersed graphene oxide solution is obtained with no visible precipitation or agglomeration. Weigh 0.8g of polyvinyl butyral (PVB) with a precision electronic balance, slowly add it to a mixing bottle containing 9.2mL of anhydrous ethanol, and stir for 30 minutes to completely dissolve the PVB. Measure 3mL of graphene oxide dispersion and add it to the completely dissolved PVB solution, continue stirring for 30 minutes to fully mix it, and obtain an electrospinning solution. The electrospinning solution is loaded into a 10cm long, 0.9mm diameter syringe for the preparation of ordered fiber scaffolds. The electrospinning parameters were set as follows: propulsion pump speed 1.5 mL / h, voltage 18 kV, and distance between needle tip and parallel electrode receiver 15 cm. Parallel oriented fiber scaffolds were collected between the electrode rods.

[0067] The prepared conductive graphene oxide electrospun membrane was cut into a circle with the same area as the hydrogel surface and laminated onto the surface of the GelMA / FPBA / osteostatin hydrogel (GF-Os) prepared above. After close adhesion, GF-Os was assembled. G Material.

[0068] Example 2

[0069] GF-Os G Characterization

[0070] Scanning electron microscopy (SEM): A FEI Scios 2HiVac scanning electron microscope (FEI, USA) was used to evaluate the hydrogel surface morphology. The samples were first freeze-dried briefly to preserve the microstructure and then gold-sprayed to enhance conductivity. SEM images were taken at an appropriate accelerating voltage to ensure high-resolution observation of hydrogel surface features.

[0071] X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR): XPS analysis was performed using an AXIS Ultra DLD spectrometer (Shimadzu, Japan), and FTIR analysis was performed using a Nicolet iS20 spectrometer (Thermo Fisher Scientific, USA). XPS was carried out under high vacuum conditions for full-spectrum and high-resolution scans to identify and quantify the elemental composition and oxidation states; the FTIR spectra were collected in the range of 4000–500 cm-1 to detect the vibrations of characteristic functional groups.

[0072] Weigh the initial weight (W0) of the weighing bracket and place it in a sealed 50 mL PBS centrifuge tube. Incubate it in a shaker at 37 °C (60 rpm). Measure the remaining weight (W t ) in the centrifuge tube at the specified time points (1, 7, 14, 21, 28 days). The degradation rate calculation formula is: Degradation rate = (W t - W0) / W0 × 100%.

[0073] Weigh the lyophilized hydrogel (W0) and place it in a sealed 50 mL PBS centrifuge tube. Observe its swelling changes in PBS at 37 °C. Record the mass of the hydrogel (W t ) at different time points (1 hour, 2 hours, 3 hours). The swelling rate calculation formula is: Swelling rate = (W t - W0) / W0 × 100%.

[0074] To determine the osteostatin release of GF-Os G at different glucose concentrations (25, 5.6, 0 mmol L-1), immerse GF-Os G in 50 mL centrifuge tubes containing the corresponding glucose solutions (10 mL). Sampling was carried out at 1, 3, 5, 10, 15, 20, 25, 30 days respectively. The amount of osteostatin released was determined using high-performance liquid chromatography (HPLC, Agilent system equipped with a Kromasil 100-5C18 chromatographic column), and the cumulative release curve was plotted.

[0075] Uniaxial tensile test (stress-strain): Use a CMT6103 testing machine (MTS Industrial Systems, China) to evaluate the tensile properties of the hydrogel. Cast the hydrogel sample into a standard dumbbell-shaped mold (compliant with ASTM standards), and carefully remove it after curing. Clamp the sample in the testing machine and apply a tensile force at a constant strain rate until fracture.

[0076] Rheological tests (modulus and viscosity): Oscillation mode (modulus measurement) and steady-state shear mode (dynamic viscosity) were performed using an MCR 302 rheometer (Anton Paar, Austria). In the oscillation mode, time sweeps were carried out by fixing the strain (within the linear viscoelastic region) and the frequency, and the storage modulus (G’) and loss modulus (G”) were recorded as functions of time. In the steady-state shear mode, the shear rate was increased logarithmically, and the corresponding viscosity values were recorded and a flow curve was plotted to characterize shear thinning or shear thickening behavior.

[0077] Conductivity test: An ST2742B automatic powder resistivity tester (four-probe method) was used to ensure accurate and reliable electrical readings. The electrospun scaffold was placed on the four-probe stage of the instrument to record the resistance value, and the conductivity was calculated from the resistivity.

[0078] Electrochemical tests: including microcurrent detection, photocurrent (IT) response, and electrochemical impedance spectroscopy (EIS), were all carried out using a CHI660E electrochemical workstation in a conventional three-electrode system. A platinum (Pt) wire was used as the counter electrode, an Ag / AgCl electrode was used as the reference electrode, and fluorine-doped tin oxide (FTO) glass coated with the photocatalyst was used as the working electrode.

[0079] Preparation of the working electrode: 5 mg of the photocatalyst was added to a 1.5 mL centrifuge tube containing 0.5 mL of ethanol and sonicated for 30 minutes to disperse it evenly. Subsequently, 10 μL of Nafion 117 (perfluorinated resin) was added and stirred well. The mixture was drop-coated onto a 1 cm × 1 cm FTO substrate and dried naturally. The edges of the coated area were painted with nail polish to protect the electrode surface from side reactions. The electrode was dried in an oven at 60 °C for 2 hours. Electrochemical tests were carried out in a 0.5 M Na2SO4 electrolyte solution, including photocurrent measurements under illumination, EIS to evaluate the charge transfer resistance, and analyses such as linear voltammetry and Tafel curves. The data were recorded using CHI660E software, and each set of measurements was repeated at least three times to ensure reproducibility.

[0080] GF-Os G Physicochemical properties of the intelligent biobattery scaffold

[0081] We prepared a GF-Os hydrogel by combining GF and G-Os components, and then assembled an electrospun graphene oxide membrane onto the surface of the GF-Os hydrogel, finally forming the intelligent biobattery GF-Os G ( Figure 9 )。GF-Os G The preparation process of the intelligent biobattery material is as Figure 2As shown in a. The GelMA prepared at a concentration of 5% exhibited good porosity. At the same time, GelMA and FPBA were dissolved in 1 mL of deionized water containing 0.1% dimethyl sulfoxide (DMSO) and mixed well. Scanning electron microscopy (SEM) showed that FPBA was completely dissolved and no visible particles were present. The resulting hydrogel maintained a porosity comparable to that of the control GelMA hydrogel, but the pore walls were thinner - this may be due to the evaporation of DMSO during the freeze-drying process. Osteostatin produced a similar effect after being dissolved in ethanol. The electrospun fibers of graphene oxide presented an ordered structure ( Figure 2 b).

[0082] To study the distribution of FPBA and osteostatin in the GelMA matrix, we performed energy-dispersive X-ray spectroscopy (EDS) analysis ( Figure 2 c). In the GF group, elemental mapping detected the characteristic F element (absent in both the G and GO groups), indicating the successful incorporation of FPBA. A uniform distribution of C, N, and O was observed in the GO group; however, since GelMA itself contains these elements, EDS data alone is not sufficient to confirm whether they are derived from osteostatin. Although the N content (19.4%) in the GO group was higher than that in the ordinary GelMA hydrogel (16.2%), considering the low atomic weight of N, the small percentage change of this element determined by the index analysis method may not be a reliable criterion and can only provide evidence for the incorporation of osteostatin. Combining SEM observations, these results preliminarily suggest that FPBA and osteostatin are present in their respective hydrogel structures, supporting subsequent electric field regulation ( Figures 10 - 13 ). To further verify the components, we subsequently performed Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) analysis.

[0083] The binding of FPBA, osteostatin, and graphene oxide film within the hydrogel scaffold was elucidated by FTIR. As Figure 2As shown in Figure d: The peak at 3273.17 cm-1 in Group G usually corresponds to N–H or O–H stretching vibrations, indicating the amino or hydroxyl groups in GelMA; the peak at 1628.03 cm-1 is attributed to C=O stretching vibration, representing the amide I band of gelatin - a key feature of GelMA. The peaks at 3278.03 cm-1 and 1630.65 cm-1 in Group GF are consistent with GelMA, indicating that the introduction of FPBA does not disrupt the basic structure of GelMA. The peaks at 836.44 cm-1 and 813.93 cm-1 originate from the C–H bending vibrations of the aromatic ring of FPBA; the peak at 522.60 cm-1 corresponds to B–O stretching vibration, which is the characteristic absorption of the boric acid group in FPBA, confirming that FPBA is encapsulated in GelMA and its basic structure remains unchanged. The spectra of Group G-Os are basically the same as those of Group G. The peak at 1533.29 cm-1 is related to N–H bending and C–N stretching vibrations and has a higher intensity due to the addition of osteostatin protein; the low-frequency peaks at 455.80 cm-1 and 417.16 cm-1 are attributed to specific vibration modes of the protein, confirming that the protein is successfully encapsulated in the GelMA matrix and the original properties of the composite material are retained. The peak at 1597.83 cm-1 in Group GF-Os corresponds to the C=C stretching vibration of the aromatic ring (indicating the presence of FPBA), and the peak at 1530.76 cm-1 originates from the amide bond of osteostatin. Compared with Group GF-Os, Group GF-Os G group retains the characteristic absorption peaks of GelMA, FPBA, and osteostatin. The newly added low-frequency peaks at 734.25 cm-1 and 674.67 cm-1 represent the characteristic absorption of carbon-oxygen or carbon-carbon structures in the graphene group. It is worth noting that the new peaks at 1090.81 cm-1 and 1014.71 cm-1 are related to C–O vibrations, indicating an interaction between graphene oxide and the composite material (the C–OH group of graphene oxide may interact with the hydroxyl group of GelMA); the peaks at 830.73 cm-1 and 812.53 cm-1 (corresponding to the C–H bending vibration of the aromatic ring) suggest a possible π–π interaction between the benzene ring structure of FPBA and graphene oxide. These results confirm that the electrospun graphene oxide film can be tightly bound to the GF-Os hydrogel through π–π interaction or hydrogen bonding.

[0084] The surface chemical changes of the hydrogel scaffold system were analyzed by XPS to verify the incorporation of the structures of FPBA, osteostatin, and graphene oxide. The pure GelMA hydrogel contains C, N, and O elements, and peaks of O=C–O, C–N, and C–C bonds can be seen in its C1s and O1s spectra. The addition of FPBA in Group GF results in peaks at 687.2 eV (C–F bond) and 191.8 eV (C–B bond). The incorporation of osteostatin in Group GO enables the detection of C=N and N–H peaks in the N1s spectrum. Group GF-Os exhibits the characteristic peaks of both Group G-Os and Group GF simultaneously, indicating the successful integration of the two components ( Figure 14)。In addition, GF-OS G In the C1s spectrum of group C, the signal of O=C–O was enhanced, confirming the successful assembly of the surface electrospun graphene oxide film ( Figure 2 e).

[0085] The swelling experiment showed that all groups of materials had good swelling rates: the freeze-dried hydrogel could rapidly swell within 30 minutes and remain stable ( Figure 15 a). The in vitro degradation experiment showed that the hydrogel degraded slowly in the initial stage (about 75% of the volume remained on the 14th day), and accelerated in the later stage (85% degraded and 15% remained on the 28th day) ( Figure 2 f). It should be noted that the main degradation component was the GelMA matrix, while the graphene oxide part usually took longer to degrade and was finally excreted in vivo. This continuous structure ensured that the hydrogel continuously generated microcurrents to regulate the immune microenvironment in the early stage of fracture healing. The drug release experiment showed that in GF-Os G the release efficiency of osteostatin gradually increased with the increase of glucose concentration ( Figure 2 g).

[0086] The mechanical properties of the hydrogel materials were characterized by stress-strain experiments. The results showed that all groups of hydrogels had good mechanical strength (able to withstand a compressive stress of about 100–125 kPa). In terms of deformation, the G, G-Os, and GF groups were slightly better than the GF-Os and GF-Os G groups (possibly because the combination of G-Os and GF reduced the deformation ability of the hydrogel), but GF-Os G still maintained a strain of more than 60% ( Figure 2 h), indicating that the assembly had no adverse effect on GF-Os G and it had excellent mechanical properties.

[0087] The storage modulus (G') and loss modulus (G") under different strain, frequency, and temperature conditions were obtained through rheological analysis. When G'>G", the hydrogel showed stronger elasticity (solid-like behavior); when G">G', it was closer to a liquid or had higher fluidity. The results showed that the hydrogels in the G, GF, and G-Os groups all showed G'>G", indicating good elasticity ( Figure 2 i). The viscosity test showed that the viscosities of the GF and G-Os groups were slightly higher than that of the G group, but with the increase of the shear rate, their viscosity decreased more slowly ( Figure 15 b).

[0088] GF-Os G Electrochemical characteristics of the intelligent biobattery

[0089] GF-Os G The system uses an electrospun graphene oxide (GO) film as the key medium for charge transfer and microcurrent generation. Figure 3a shows its spontaneous microcurrent generation process. In this study, the electrochemical properties of GO films with different concentrations were optimized: a complete Go2 film was prepared with 0.2% (w / v) GO; the Go3 film exhibited a loose and porous structure at a concentration of 0.3%; and a Go4 film could not be formed at a concentration of 0.4%. Previous studies have shown that a loose and porous structure is beneficial for cell proliferation and migration. Therefore, GO films with better performance were screened through electrochemical characterization.

[0090] Cyclic voltammetry (CV) was used to evaluate the capacitive behavior of the films. The results showed that Go3 had a larger capacitive area, a higher density of active sites, and a larger specific surface area than Go2 ( Figure 3 b and Figure 16 ), laying the foundation for efficient charge transfer. Double-layer capacitance (Cdl) measurements showed that Go3 had a higher capacitance ( Figure 3 c), indicating better charge storage and release efficiency. Linear sweep voltammetry (LSV) curves showed that Go3 had better electrocatalytic activity and efficiency for the hydrogen evolution reaction (HER) ( Figure 3 d), which may be attributed to its optimized microstructure and chemical composition. The overpotential of Go3 at a current density of 10 mA / cm 2 was 368 mV (565 mV for Go2) ( Figure 3 e), and the Tafel slope (43.2 mV / dec vs. 63.5 mV / dec for Go2) indicated faster electron transfer kinetics ( Figure 3 f). The smaller semicircle diameter of Go3 in the EIS Nyquist plot directly reflected lower charge transfer resistance and higher electron mobility ( Figure 3 g).

[0091] In the 2400-second chronoamperometry test at a constant overpotential, Go3 maintained a stable transient current density; after 1000 consecutive LSV cycles, the curve showed no obvious shift, confirming its excellent electrochemical stability ( Figure 17 a, b). The 2400-second photocurrent stability test showed that the current density of Go3 fluctuated very little ( Figure 18 a, b). Conductivity measurements showed that the conductivity increased exponentially when the concentration of Go3 increased by 0.1% ( Figure 3 h), providing a basic guarantee for microcurrent generation and transfer within the material.

[0092] The surface charge changes of GF and G-Os hydrogels at different glucose concentrations were evaluated by measuring the Zeta potential (ζ). In a PBS environment, the ζ of the GF hydrogel was approximately -6.38 mV, and that of G-Os was approximately +24.96 mV; when the glucose concentration increased to 11.1 mM, the ζ of GF decreased to -39.7 mV, and that of G-Os increased to +34.97 mV (the potential difference was approximately 74.67 mV); at 25 mM glucose, the ζ of GF reached -50.1 mV, and that of G-Os was +36.0 mV (the potential difference was 86.1 mV).Figure 19 a-d) showed that elevated glucose could enhance the negative charge of GF and the positive charge of G-Os, expanding the potential difference between the two. Based on the electrical characterization, the Go3 film was preferably selected as the experimental group material.

[0093] Investigate GF-Os G Current transmission between the GO end and the GF end of the material: An initial microcurrent of approximately 1 μA was detected at the GF end in the high-glucose solution ( Figure 3 i); after inserting an insulating film between GF and G-Os, the current at the GF end tended to zero, confirming that the charge transferred from the G-Os segment to the GF end. At the 2nd, 3rd, and 4th weeks, the current at the GF end decreased to 0.4 μA, 0.2 μA, and nearly zero, respectively ( Figure 20 a-c). Figure 21 It showed that no microcurrent was generated under normal physiological conditions, indicating that the material had a high-glucose environment responsiveness. This continuous microcurrent generation was synchronized with the early stage of fracture healing and could regulate the immune microenvironment through electrotherapy.

[0094] Biocompatibility and ROS scavenging ability

[0095] The biocompatibility of the self-powered hydrogel scaffold was evaluated by live / dead cell staining. In all groups, the live cells (green fluorescence) were in good condition, accounting for more than 95%, and only a very small number of dead cells (red fluorescence) were observed, indicating that each group had high cell activity ( Figure 22 a,b). To analyze the proliferation ability of BMSCs, the cells were co-cultured with the scaffold for 5 days and CCK-8 assays were performed on the 1st, 3rd, and 5th days. The results showed that the cells in all groups maintained strong proliferation ability at the three time points, and the cells in the GF-Os G group proliferated significantly better than those in other groups (P < 0.05), indicating that electrical stimulation (ES) could promote BMSC proliferation ( Figure 23 ). The live / dead staining and CCK-8 experiments together demonstrated that GF-Os G had excellent biocompatibility. Combining its good mechanical and electrical properties, it was very suitable for subsequent biological experiments.

[0096] Further study the ROS scavenging ability of GF-Os G . The pro-inflammatory response in the diabetic microenvironment led to a continuous increase in the ROS level in the early stage of fracture healing. After co-culturing the cells with the scaffold for one week, ROS detection was performed. The results showed that the proportion of ROS-positive cells (green fluorescence) in the GF-Os G group was the lowest ( Figure 24 a), and the quantitative analysis of fluorescence intensity showed that the ROS level in the GF-Os G group was significantly lower than that in other groups (P < 0.05), while there was no significant difference between the other groups (P > 0.05) ( Figure 24 b). Flow cytometry confirmed that compared with GF-Os GThe ROS level in co-cultured BMSC cells was significantly lower than that in other groups (P<0.05). Figure 25 ) These results indicate that the ROS scavenging effect of the scaffold is not due to the hydrogel material itself, but is mediated by the microcurrent generated by the GF-Os G system.

[0097] Example 3

[0098] Macrophage polarization analysis

[0099] Bone marrow-derived macrophages (BMDMs) were extracted from the femurs of 6-week-old C57BL / 6 mice. Macrophage polarization was evaluated by immunostaining and flow cytometry. BMDMs were seeded in 12-well plates at a density of 4×104 cells / well and cultured for 12 hours. Cells were induced with 100 ng / mL LPS and 25 mM glucose (high glucose) and co-cultured with different scaffolds. Immunofluorescence analysis (observed by inverted fluorescence microscopy) was performed using inducible nitric oxide synthase (CD86; 1:500, ABclonal) and CD206 (1:500, ABclonal) markers. The expression levels of CD86 and CD206 were quantified by flow cytometry. In addition, the expression of inflammation-related genes was detected by real-time quantitative PCR (qRT-PCR). Total RNA was extracted using TRIzol reagent, cDNA was synthesized using the PrimeScript RT kit, and the primer sequences for qRT-PCR analysis are shown in Table 1.

[0100] Table 1 Primer sequences for murine BMDMs

[0101]

[0102]

[0103] Regulation of macrophage polarization phenotype

[0104] The diabetic microenvironment exacerbates the early inflammatory response after fracture, and exogenous biomaterials may exacerbate local inflammation after implantation. Therefore, the ability of biomaterials to regulate macrophage polarization is crucial. Increasing the M2 / M1 macrophage ratio can effectively improve the inflammatory microenvironment and promote bone regeneration. The biocompatibility of the scaffold with macrophages was evaluated by live / dead staining: macrophages were mainly green fluorescent (live cells) in all groups, and the proportion of live cells exceeded 95% Figure 26 a, b), indicating that the scaffold has excellent biocompatibility with macrophages. Histological evaluation of the main organs (heart, liver, spleen, lung, kidney) in animal experiments showed no pathological changes or tissue damage.

[0105] The effect of the composite scaffold on macrophage polarization was explored by immunofluorescence staining. As Figure 4 shown in a, GF-OsG The expression of CD86 (M1 marker) in the group was significantly lower than that in the G, GF, G-Os, and GF-Os groups, while the expression of the M2 marker CD206 was significantly upregulated. Quantitative analysis of CD86 and CD206 expression by flow cytometry ( Figure 4 b) showed that the proportion of M1 macrophages in the GF-Os G group decreased, and the proportion of M2 increased to 24.8%. RT-qPCR was used to detect the gene expression of inflammatory factors ( Figure 4 c-f) indicated that the anti-inflammatory factors (IL-10, TGF-β) in the GF-Os G group were significantly increased, while the pro-inflammatory factors (TNF-α, iNOS) were significantly decreased. Western blot ( Figure 4 g-h) showed that the GF-Os G group significantly downregulated the M1 markers (CD86, iNOS) and upregulated the M2 markers (CD206, ARG1). ELISA analysis ( Figure 4 i) further confirmed that GF-Os G reduced the levels of pro-inflammatory factors (TNF-α, IL-6) and increased the anti-inflammatory factors (TGF-β, IL-10). In summary, GF-Os G scaffold effectively regulated the immune microenvironment by promoting M2 polarization and promoted bone regeneration.

[0106] Example 4

[0107] Vascularization evaluation

[0108] Cell migration experiment: Human umbilical vein endothelial cells (HUVECs) were seeded in the upper chamber of a 24-well Transwell plate, and different scaffolds were placed in the lower chamber. After co-culture for 12 hours, the cells were fixed with 4% paraformaldehyde (PFA), stained with crystal violet (Beyotime) for 15 minutes, and rinsed three times with PBS. The non-migrated cells on the surface of the upper membrane were gently wiped off with a cotton swab, and the images of the migrated cells were taken with an optical microscope. The migration rate was quantified using ImageJ software.

[0109] Angiogenesis experiment: HUVECs were seeded in a 24-well plate with growth factor-reduced Matrigel (BD Biosciences) to evaluate the formation of tubular structures. After co-culture with different scaffolds for 4 hours, the tubular structures were observed with an optical microscope and quantified using ImageJ software. Immunofluorescence staining of the angiogenesis marker CD31 (1:200, ABclonal) was performed as described above.

[0110] GF-Os G Effect of the intelligent biobattery on in vitro angiogenesis

[0111] The effect of the scaffold on the migration ability of HUVECs was evaluated by Transwell migration assay. After co-culture for 12 h, crystal violet staining showed that the number of cells migrated to the lower chamber in the GF-Os G group was significantly more than that in the other four groups ( Figure 27 a), and quantitative analysis confirmed that its migration ability was significantly enhanced (P<0.05) ( Figure 27 b).

[0112] Tube formation assay showed that HUVECs in the GF-Os G group formed extensive tubular networks ( Figure 28 a), and the total tube length quantification indicated that its angiogenesis-promoting ability was significantly better than that of other groups ( Figure 28 b). CD31 immunofluorescence staining showed that the CD31 expression and fluorescence intensity of HUVECs in the GF-Os G group were the highest ( Figure 29 a, b), further confirming its angiogenesis-promoting effect.

[0113] Example 5

[0114] Osteogenic differentiation evaluation

[0115] Bone marrow mesenchymal stem cells (BMSCs) were co-cultured with different scaffolds in high-glucose α-MEM osteogenic induction medium (containing 10% fetal bovine serum, 1% penicillin-streptomycin, 100 nM dexamethasone, 50 μg / mL L-ascorbic acid, 10 mM β-glycerophosphate). The glucose concentration was adjusted to 35 mmol / L. After culturing for 7 days and 21 days, the cells were fixed with 4% PFA, and the alkaline phosphatase (ALP) activity and mineral deposition were evaluated by staining with BCIP / NBT working solution (Beyotime) and alizarin red S (ARS; Beyotime), respectively. The ALP activity was further quantified using an ALP detection kit (Beyotime), and semi-quantitative analysis of ARS staining was performed after dissolving the stained substances with hydrochloric acid.

[0116] Immunofluorescence staining of type I collagen α1 chain (COL1A1): After induction for 7 days, the cells were fixed with 4% PFA for 15 min, permeabilized with 0.1% Triton X-100 for 20 min, and blocked with 1% BSA for 40 min. The cells were incubated with COL1A1 primary antibody (1:500, ABclonal) at 4°C overnight, and then incubated with fluorescently labeled secondary antibody (1:500, ABclonal). The cell nuclei were stained with DAPI (Thermo Fisher), and images were acquired using an inverted fluorescence microscope (Zeiss), and the fluorescence intensity was quantified using ImageJ software.

[0117] qRT-PCR analysis of osteogenesis-related genes: Total RNA was extracted using TRIzol reagent after induction for 7 days, reverse-transcribed into cDNA using a PrimeScript RT kit, and the qRT-PCR primer sequences are shown in Table 2.

[0118] Table 2 Primer sequences of mouse BMSCs

[0119]

[0120] Osteoclast differentiation evaluation

[0121] Bone marrow-derived macrophages (BMDMs) were isolated from the femurs of 6-week-old C57BL / 6 mice and co-cultured with different scaffolds in high-glucose medium containing 30 ng / mL macrophage colony-stimulating factor (M-CSF; ABclonal Technology Co., Ltd.) and 50 ng / mL receptor activator of nuclear factor-κB ligand (RANKL; Servicebio) to promote osteoclast differentiation. After 7 days of culture, the cells were fixed with 4% PFA for 30 minutes and stained with a tartrate-resistant acid phosphatase (TRAP) staining kit (Sigma-Aldrich). TRAP-positive cells with more than three nuclei were identified as osteoclasts under a microscope. The formation of mature osteoclasts was further verified by F-actin immunofluorescence staining (DAPI counterstaining of nuclei). Images were taken with a fluorescence microscope, and the total area of the TRAP-positive region and the number of osteoclasts were quantified using ImageJ software. The expression of osteoclast-related genes was evaluated by qRT-PCR, and the primer sequences are shown in Table 3.

[0122] Table 3 Primer sequences of mouse osteoclasts

[0123]

[0124] GF-Os G Regulation of in vitro osteogenesis and osteoclast differentiation by intelligent biobattery

[0125] Osteogenic differentiation: After co-culturing BMSCs with scaffolds for 7 days, ALP staining and activity quantification were performed. The ALP staining intensity of the GF-Os G group was the strongest ( Figure 5 a, b), and ARS staining at 21 days showed the most calcium nodule deposition ( Figure 5 c, d). COL1 immunofluorescence showed the strongest expression in the GF-Os G group ( Figure 30 a, b), and RT-qPCR detected a significant upregulation of the expression of osteogenic genes (Alpl, Bglap, Col1a1, Runx2) ( Figure 5 e). Western blot confirmed that the expression of COL1A1 and RUNX2 in the GF-Os G group was significantly higher than that in other groups ( Figure 31 a - c).

[0126] Inhibition of osteoclast differentiation: Network pharmacology analysis screened out core targets such as EP300, SIRT1, KAT2B, HDAC4, etc. (Table 4 andFigure 24 a-d). Molecular docking showed that osteostatin had the lowest binding energy with HDAC4 (Table 5 and Figure 33 ). TRAP staining showed that osteoclast formation in the GF-Os G , GF-Os, and G-Os groups was significantly inhibited ( Figure 5 f, g). SEM observation of bone resorption lacunae showed that the area of the GF-Os G group was the smallest ( Figure 5 h, i). F-actin immunofluorescence showed that the formation of actin rings in the GF-Os G group was the least ( Figure 5 j, k). RT-qPCR detection showed that the expression of osteoclast-related genes (Mmp9, Nfatc1, Fos, Acp5) was significantly downregulated ( Figure 5 l). Western blot showed that the expression of NFATC1 and MMP9 in the GF-Os G group was the lowest ( Figure 34 a-c).

[0127] Table 4 Possible target proteins for osteostatin inhibition of osteoclast differentiation

[0128]

[0129]

[0130] Table 5 Binding ability of osteostatin with core target genes

[0131]

[0132] Example 6

[0133] Evaluation of bone regeneration in diabetic rats in vivo

[0134] Diabetic rat cranial defect model: All animal experiments were approved by the Animal Ethics Committee of Soochow University (approval number: SUDA20240913A07). After 60 male 8-week-old SD rats were fed a high-sugar and high-fat diet for 1 month, they were intraperitoneally injected with streptozotocin (STZ; Sigma-Aldrich, dissolved in pH 4.5 citrate buffer, dose 60 mg / kg) to induce diabetes. Rats with blood glucose > 16.7 mmol / L were determined to have diabetes. The diabetic rats were randomly divided into 6 groups: NC (negative control) group, G group, GF group, G-Os group, GF-Os group, and GF-Os G group. Under anesthesia and sterile conditions, a 5-mm critical-sized bone defect was created on both sides of the rat skull and the corresponding scaffolds were implanted. The rats were sacrificed at 4 and 8 weeks after surgery, and the skulls were taken for Micro-CT and histological analysis. No signs of pain or discomfort were observed during or after the surgery and throughout the study.

[0135] Micro-CT evaluation: The skull was scanned using a SkyScan 1176 Micro-CT system (Bruker, USA). Bone regeneration was quantified by bone volume fraction (BV / TV, %), trabecular bone number (Tb.N, 1 / mm), and bone mineral density (BMD, g / cm 3 ).

[0136] Histological analysis: The specimens were decalcified with 10% ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich) for 1 month, embedded in paraffin, and sectioned (6 μm thick). Hematoxylin-eosin (H&E), Masson's trichrome staining, immunohistochemistry, and TRAP staining were performed according to standard procedures. The immune microenvironment was analyzed by immunohistochemistry for tumor necrosis factor-α (TNF-α; 1:500, ABclonal) and transforming growth factor-β (TGF-β; 1:500, ABclonal) 4 weeks after implantation.

[0137] Evaluation of bone regeneration in a rat cranial defect model in vivo

[0138] The scaffolds were implanted into the cranial defects of diabetic rats. Micro-CT showed that the amount of new bone formation (BV / TV) in the GF-Os G group was significantly higher than that in other groups at 4 and 8 weeks ( Figure 6 b), and the bone mineral density (BMD) and trabecular bone parameters (Tb.N, Tb.Th) were also significantly improved ( Figure 6 c). H&E and Masson staining showed that the GF-Os G group had the most abundant new bone tissue and the most collagen deposition in the defect area ( Figure 6 d). Histological evaluation of major organs showed no abnormalities ( Figure 35 ).

[0139] Immunohistochemical analysis showed that TGF-β expression was upregulated and TNF-α expression was downregulated in the GF-Os G group ( Figure 7 a, b), the positive areas of VEGF and CD31 were significantly increased ( Figure 7 c - e), the COL1 deposition and RUNX2 expression were the highest ( Figure 7 f - h), and the number of TRAP-positive cells was the least ( Figure 7 i, j), indicating that it significantly inhibited osteoclastic activity.

[0140] Example 7

[0141] Molecular mechanism and Western blot analysis

[0142] BMSCs were cultured under two conditions: containing GF-OS GThe hyperglycemic environment of the scaffold (experimental group) and the hyperglycemic environment without scaffold (control group). Samples were collected immediately after culture and snap-frozen in liquid nitrogen. The frozen samples were pulverized to ensure repeatability and consistency.

[0143] qRT-PCR: Total RNA was extracted using TRIzol reagent (Invitrogen, USA), cDNA was synthesized using the Takara reverse transcription kit, and qRT-PCR was performed using the Thermo Maxima SYBR Green / ROX qPCR premix, with β-actin as the internal reference.

[0144] Proteomics analysis: Proteomics analysis was performed on BMSCs co-cultured with the hydrogel (3 biological replicates per group). After protein extraction, identification and quantification were performed by reversed-phase high-performance liquid chromatography (Bio-Rad, USA). The mass spectrometry data were analyzed using MaxQuant / Andromeda software (v1.3.0.5), and bioinformatics analysis was performed using DAVID, STRING, Cytoscape, and OmicStudio tools. Differentially expressed proteins were screened with a significance threshold of p < 0.05.

[0145] Immunoblotting: BMSCs were lysed with ice-cold lysis buffer [150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 20 mM Tris-HCl (pH 7.5)] (containing protease and phosphatase inhibitors). The extracted proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). After blocking the membrane with 5% non-fat milk powder, it was incubated with primary antibodies overnight at 4°C: phosphorylated p38 MAPK (Thr180 / Tyr182; CST, 4511), p38 MAPK (CST, 54470), phosphorylated ERK1 / 2 (Thr202 / Tyr204; Affinity, AF1015), ERK1 / 2 (Affinity, AF0155), GPX4 (1:2000, Affinity, DF6701), β-actin (CST, 4970). Subsequently, it was incubated with a horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch) for 1.5 hours at room temperature. Protein bands were developed using a chemiluminescence detection system (Vilber).

[0146] 2.9. Statistical analysis

[0147] Data are presented as mean ± standard deviation (at least 3 independent experiments). One-way analysis of variance (ANOVA) was used for differences among multiple groups, and two-tailed Student's t-test was used for differences between groups. SPSS 20.0, Microsoft Excel, and GraphPad Prism 9.0 were used for plotting. Significance was marked as *P < 0.05, **P < 0.01, ***P < 0.001.

[0148] Exploration of the molecular mechanism of the BMSC osteogenic pathway

[0149] RNA sequencing (RNA-seq) showed significant differences in gene expression between the GF-Os G group and the control group ( Figure 36 a,b). Gene Set Enrichment Analysis (GSEA) suggested that GF-Os G significantly affected pathways such as growth factor binding and electron transfer activity ( Figure 37 a,b). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed significant differences in the MAPK signaling pathway, Cushing's syndrome, etc. ( Figure 8 c,d; Figure 38 a,b). Gene Ontology (GO) analysis indicated that the differentially expressed genes were involved in cell adhesion, inflammatory response, and positive regulation of the MAPK cascade ( Figure 8 e,f). Western blot confirmed that GF-Os G activated the ERK / P38-GPX4 pathway: the expression of p-ERK, p-p38, and GPX4 was upregulated ( Figure 8 h-k), and the inhibitor could reverse this effect, verifying its antioxidant and osteogenic-promoting effects through the MAPK pathway ( Figure 8 g).

Claims

1. A preparation method of an intelligent biological battery for diabetic bone defect regeneration, characterized in that, The preparation method includes: assembling the methylacrylated gelatin hydrogel scaffold loaded with tetrafluorobenzeneboronic acid and the methylacrylated gelatin hydrogel scaffold loaded with osteostatin respectively, and then tightly integrating the conductive graphene oxide electrospun membrane with the hydrogel scaffold through a self-assembly process to obtain an intelligent biobattery for diabetic bone defect regeneration.

2. The preparation method of the intelligent biobattery for diabetic bone defect regeneration according to claim 1, wherein, The self-assembly process includes: placing the methylacrylated gelatin hydrogel scaffold loaded with tetrafluorobenzeneboronic acid and the methylacrylated gelatin hydrogel scaffold loaded with osteostatin on both sides of a separator membrane respectively; laminating the conductive graphene oxide electrospun membrane onto the surfaces of the methylacrylated gelatin hydrogel scaffold loaded with tetrafluorobenzeneboronic acid and the methylacrylated gelatin hydrogel scaffold loaded with osteostatin, and obtaining an intelligent biobattery for diabetic bone defect regeneration after tightly fitting and assembling.

3. The preparation method of the intelligent biobattery for diabetic bone defect regeneration according to claim 1, characterized in that, The preparation method of the conductive graphene oxide electrospun membrane includes: slowly adding polyvinyl butyral into absolute ethanol, fully dissolving it, adding graphene oxide dispersion liquid, and fully stirring and mixing to obtain an electrospinning solution, and preparing the conductive graphene oxide electrospun membrane.

4. The preparation method of the intelligent biobattery for diabetic bone defect regeneration according to claim 3, characterized in that, The preparation method of the graphene oxide dispersion liquid includes: adding graphene oxide powder into deionized water, fully mixing, and performing ultrasonic treatment under ice-water bath conditions to obtain a uniformly dispersed graphene oxide solution.

5. The preparation method of the intelligent biobattery for diabetic bone defect regeneration according to claim 1, characterized in that, The preparation method of the methylacrylated gelatin hydrogel scaffold loaded with tetrafluorobenzeneboronic acid includes: dissolving freeze-dried methylacrylated gelatin and tetrafluorobenzeneboronic acid in an aqueous solution of dimethyl sulfoxide, fully mixing, adding a photoinitiator into the mixed solution, stirring until completely dissolved, and completing photocrosslinking after ultraviolet irradiation to obtain the methylacrylated gelatin hydrogel scaffold loaded with tetrafluorobenzeneboronic acid.

6. The preparation method of the intelligent biobattery for diabetic bone defect regeneration according to claim 1, characterized in that, The preparation method of the methylacrylated gelatin hydrogel scaffold loaded with osteostatin includes: dissolving freeze-dried methylacrylated gelatin and osteostatin in PBS and fully mixing, adding a photoinitiator into the mixed solution, stirring until completely dissolved, and completing photocrosslinking after ultraviolet irradiation to obtain the methylacrylated gelatin hydrogel scaffold loaded with tetrafluorobenzeneboronic acid.

7. An intelligent biobattery for diabetic bone defect regeneration, characterized in that, Prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the intelligent biobattery for diabetic bone defect regeneration according to claim 7 in the preparation of a bone immune regulation and repair material.

9. Use of the intelligent biobattery for diabetic bone defect regeneration according to claim 7 in reducing the intracellular ROS level.

10. Use of the intelligent biobattery for diabetic bone defect regeneration according to claim 7 in increasing the ratio of M2 / M1 macrophages.

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