A bioactive scaffold and its preparation method and use

Abstract

This invention belongs to the field of biomaterials technology, specifically relating to a bioactive scaffold, its preparation method, and its applications. This invention incorporates functionalized mesoporous silica nanoparticles loaded with aminoguanidine into the skin secretions of the giant salamander and polycaprolactone electrospun fibers to construct a bioactive composite scaffold (SPMA). This scaffold enables the sustained release of AG and osteogenic factors, effectively inhibiting free radical generation, AGEs toxicity, and inflammatory responses. Furthermore, it significantly improves the survival rate, osteogenic differentiation, and mineralization capacity of bone marrow mesenchymal stem cells in a high-glucose microenvironment. In vivo experiments further demonstrate that in a streptozotocin-induced diabetic mouse model of skull defects, the SPMA scaffold can alleviate local inflammation, promote angiogenesis, and significantly accelerate new bone formation and defect repair.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a bioactive scaffold, its preparation method, and its applications. Background Technology

[0002] Diabetes mellitus is a chronic metabolic disease characterized by persistently high blood sugar. Compared to non-diabetic individuals, diabetic patients have a significantly increased risk of fractures and markedly impaired bone healing. Therefore, there is an urgent need for effective bone regeneration therapies in this metabolically impaired population, and innovative treatment strategies to accelerate bone repair in diabetic patients need to be developed.

[0003] Impaired bone regeneration in diabetes is primarily attributed to a harmful wound microenvironment characterized by excessive oxidative stress, chronic inflammation, and impaired angiogenesis. Hyperglycemia can induce excessive production of free radicals and advanced glycation end products (AGEs), thereby exacerbating inflammatory responses, impairing osteoblast function, and disrupting endothelial cell activity, leading to inhibited angiogenesis. Traditional tissue-engineered scaffolds, such as those based on polycaprolactone (PCL) or bioactive ceramics, often struggle to effectively address these multiple challenges, exhibiting poor cell adhesion, persistent inflammation, and insufficient delivery of bioactive factors in diabetic models. While some strategies integrating growth factors (such as BMP-2 or VEGF) or anti-inflammatory drugs have shown promise, issues such as burst release, high cost, and potential immunogenicity limit their clinical translation.

[0004] Therefore, developing multifunctional biological scaffolds that can simultaneously regulate the diabetic microenvironment through sustained antioxidant, anti-inflammatory, and regenerative effects remains an urgent problem to be solved. Summary of the Invention

[0005] This invention provides a novel electrospun composite scaffold (SPMA), in which SSAD derived from the skin secretions of the giant salamander is loaded onto aminoguanidine (AG)-functionalized mesoporous silica nanoparticles (MSN) and integrated into a nanofiber matrix to obtain a bioactive scaffold with a nanofiber structure.

[0006] In a first aspect, the present invention provides a bioactive scaffold obtained by integrating giant salamander skin secretions and mesoporous silica nanoparticles loaded with aminoguanidine into polycaprolactone; and the bioactive scaffold has a nanofiber structure. Among them, the skin secretions of the giant salamander are loaded in mesoporous silica nanoparticles containing aminoguanidine; The mesoporous silica nanoparticles loaded with aminoguanidine are obtained by grafting aminoguanidine onto the surface of mesoporous silica nanoparticles. The mass ratio of the aminoguanidine to the mesoporous silica nanoparticles is 5:1.

[0007] In some embodiments, the bioactive scaffold has a nanofiber structure with a diameter of 300 nm to 800 nm.

[0008] In some implementations, the giant salamander skin secretions are provided by freeze-dried giant salamander skin secretion powder.

[0009] In some embodiments, the mass ratio of the freeze-dried powder of giant salamander skin secretions to mesoporous silica nanoparticles loaded with aminoguanidine is 20:1 to 100:1.

[0010] In some preferred embodiments, the mass ratio of the freeze-dried powder of giant salamander skin secretions to mesoporous silica nanoparticles loaded with aminoguanidine is 40:1.

[0011] In some embodiments, the mass ratio of polycaprolactone to freeze-dried powder of giant salamander skin secretions is 4:1.

[0012] In a second aspect, the present invention provides a method for preparing the aforementioned bioactive scaffold, the method comprising: (1) Polycaprolactone and lyophilized powder of giant salamander skin secretions were dissolved in hexafluoroisopropanol (HFIP) and mixed evenly to obtain a mixed solution; (2) Add mesoporous silica nanoparticles loaded with aminoguanidine to the mixed solution and mix them evenly to obtain a spinning solution; (3) The spinning solution is electrospun to obtain the bioactive scaffold.

[0013] In some embodiments, the mass-to-volume ratio of the polycaprolactone to the hexafluoroisopropanol is 1:10.

[0014] In some embodiments, the mass-to-volume ratio of the freeze-dried powder of the giant salamander skin secretions to the hexafluoroisopropanol is 1:40.

[0015] The main reasons for choosing HFIP as the optimal solvent in this invention are as follows: 1. Multi-component co-solubility: Giant salamander skin secretions contain abundant natural protein components, which are prone to denaturation or irreversible aggregation in traditional hydrophobic solvents such as chloroform or dichloromethane. HFIP has the outstanding ability to simultaneously dissolve hydrophobic synthetic polymers (PCL) and amphiphilic / hydrophilic natural proteins (SSAD) into a completely homogeneous and stable spinning solution without phase separation.

[0016] 2. Spinability and Morphology Control: HFIP exhibits high volatility and excellent dielectric constant. In preliminary screening experiments, other alternative solvents often led to instability in the Taylor cone, frequent needle blockage due to nanoparticle aggregation, or severe beaded fiber morphology. HFIP, however, consistently ensures the formation of a smooth, continuous, and bead-free nanofiber framework with uniform diameter distribution, which is crucial for stable mechanical properties.

[0017] In some embodiments, the mass ratio of aminoguanidine to mesoporous silica nanoparticles in the aminoguanidine-loaded mesoporous silica nanoparticles is 5:1.

[0018] In some embodiments, the method for preparing the aminoguanidine-loaded mesoporous silica nanoparticles includes: Mesoporous silica nanoparticles were dispersed in anhydrous ethanol containing aminoguanidine, and after ultrasonic treatment, they were mixed evenly at room temperature; after centrifugation and washing, mesoporous silica nanoparticles loaded with aminoguanidine were obtained. The mass ratio of the aminoguanidine to the mesoporous silica nanoparticles is 5:1; The mass-to-volume ratio of the aminoguanidine to the anhydrous ethanol is 1:25.

[0019] In some embodiments, the electrospinning conditions are: temperature of 25±2°C, relative humidity of 40%±5%, and voltage parameter set to 18 kV; The spinning solution is propelled through a needle with an inner diameter of 19 G at a constant flow rate of 0.0013 m / s into the roller collector of the electrospinning system. The roller collector rotates at 800 rpm, and the vertical receiving distance from the needle tip to the surface of the roller collector is set to 15 cm.

[0020] In some preferred embodiments, the electrospinning includes the following steps: 1) After drawing the spinning solution with a syringe, a needle with an inner diameter of 19G is then attached to the front end of the syringe; the filled syringe is fixedly installed on a micro-injection pump, and the operating parameters of the micro-injection pump are adjusted so that the spinning solution is propelled outward through the needle at a constant flow rate of 0.0013 m / s. 2) Provide an electrospinning system consisting of a micro-injection pump, a power supply, and a roller collector; fix the syringe with the needle installed horizontally so that the needle tip faces the roller collector, the surface of the roller collector is covered with aluminum foil, and the vertical receiving distance from the needle tip to the surface of the aluminum foil is set to 15 cm; connect the positive output terminal of the power supply to the needle and the negative terminal of the power supply to the bearing of the roller collector to build a high-voltage electrostatic field between the needle tip and the aluminum foil roller; 3) In an environment with a temperature of 25±2°C and a relative humidity of 40%±5%, start the electrospinning system; set the applied voltage parameter to 18 kV and control the rotation speed of the roller collector to 800 rpm; under the action of electrostatic repulsion, the spinning solution droplets at the tip of the needle form Taylor cones and are ejected as the solvent evaporates in the space, and the nanofibers are uniformly deposited on the rotating aluminum foil surface. Continuous spinning is carried out for 10 hours to obtain a bioactive scaffold of a set thickness.

[0021] In a third aspect, the present invention provides the use of the aforementioned bioactive scaffold, or the bioactive scaffold obtained by the aforementioned preparation method, in the preparation of a medicament for repairing diabetic bone defects.

[0022] In some embodiments, the drug is used to scavenge free radicals and / or regulate inflammation.

[0023] In some embodiments, the drug is used to promote angiogenesis and / or bone regeneration-related cell migration.

[0024] This invention provides a multifunctional electrospun composite bioactive scaffold (SPMA), which constructs a bioactive scaffold with a nanofiber structure by integrating SSAD-loaded AG-grafted MSN into a PCL nanofiber matrix; wherein, the skin secretions of the giant salamander are loaded in mesoporous silica nanoparticles containing aminoguanidine, and the aminoguanidine-loaded mesoporous silica nanoparticles are obtained by grafting aminoguanidine onto the surface of the mesoporous silica nanoparticles, and the mass ratio of aminoguanidine to the mesoporous silica nanoparticles is 5:1.

[0025] This bioactive scaffold simultaneously targets the core pathological features of diabetic bone defects—persistent oxidative stress, chronic inflammation, and impaired angiogenesis / osteogenesis. The scaffold achieves controlled release of naturally derived SSAD and the intrinsic antioxidant / anti-inflammatory activity of AG, effectively reprogramming the harmful diabetic microenvironment. It demonstrates the synergistic effect of combining bioactive natural extracts (SSAD) with simple, scalable AG functionalization in a nanofiber delivery system, offering cost-effectiveness, efficiency, and no growth factor dependence, overcoming the limitations of traditional scaffolds in metabolically damaged tissues. Its excellent biocompatibility, lack of systemic toxicity, and robust wound salvage in a rigorous diabetic model highlight the clinical translational potential of SPMA in diabetic bone repair, diabetic wound healing, and other hyperglycemia-related regenerative challenges. Attached Figure Description

[0026] Figure 1 The preparation process of freeze-dried powder of giant salamander skin secretions is shown.

[0027] Figure 2 A schematic diagram of PCL / SSAD mixtures with different mass ratios (pure PCL, 10:1, 4:1 and 2:1) is shown.

[0028] Figure 3 This is a schematic photograph of the spinning process and the resulting flexible membrane. The scale bar is 1 μm.

[0029] Figure 4 Representative SEM images of PCL / SSAD scaffolds with different mass ratios (pure PCL, 10:1, 4:1, and 2:1) are shown. Scale bar is 5 μm.

[0030] Figure 5 Histograms of diameter distribution for PCL / SSAD stents with different mass ratios (pure PCL, 10:1, 4:1, and 2:1) are shown.

[0031] Figure 6 FTIR spectra of PCL / SSAD stents with different mass ratios (pure PCL, 10:1, 4:1 and 2:1) are shown.

[0032] Figure 7 The cumulative in vitro SSAD release curves of PCL / SSAD stents with different mass ratios (pure PCL, 10:1, 4:1 and 2:1) are shown.

[0033] Figure 8 The results of surface water contact angle measurements for PCL / SSAD supports with different mass ratios (pure PCL, 10:1, 4:1 and 2:1) are shown.

[0034] Figure 9 Live-cell fluorescence images of HUVECs cultured on nanofiber scaffolds for 1 and 3 days are shown. Calcein-AM, green, scale bar 50 μm.

[0035] Figure 10 Experimental images of HUVECs tube formation with different nanofiber scaffolds are shown, with a scale bar of 1.4 mm.

[0036] Figure 11 The qRT-PCR analysis of CD31 and VEGF gene expression in HUVECs with different nanofiber scaffolds is shown.

[0037] Figure 12 The quantitative data of CCK-8 cell proliferation on nanofiber scaffolds with different SSAD loadings (1 day, 3 days, 5 days) are shown.

[0038] Figure 13 The live and dead staining of MSCs on different nanofiber scaffolds is shown. Calcein-AM / PI, green / red, scale bar is 100 μm. Among them, PCL is a nanofiber scaffold prepared by pure PCL, and SSAD is a nanofiber scaffold prepared by PCL / SSAD mass ratio of 4:1.

[0039] Figure 14 The results of ALP staining on day 7 and ARS staining on day 21 for different nanofiber scaffolds are shown. Among them, PCL is a nanofiber scaffold prepared from pure PCL, and SSAD is a nanofiber scaffold prepared from PCL / SSAD at a mass ratio of 4:1.

[0040] Figure 15 Different nanofiber scaffolds are shown. OCN , OPN , RUNX2 qRT-PCR quantification of gene expression. NC represents the control nanofiber scaffold; PCL represents a nanofiber scaffold prepared from pure PCL; and SSAD represents a nanofiber scaffold prepared from PCL / SSAD at a mass ratio of 4:1. p <0.05,** p <0.01, *** p <0.001. Data are expressed as mean ± SD.

[0041] Figure 16 The activity of CCK-8 cells in RAW264.7 cells treated with different concentrations of AG (0 μM to 100 μM) is shown.

[0042] Figure 17 The study demonstrated that AG dose-dependent inhibition of LPS-induced NO release was achieved.

[0043] Figure 18 The scavenging activity of AG (20 nM to 400 nM) against ABTS radicals was demonstrated.

[0044] Figure 19 The scavenging activity of AG (20 nM to 400 nM) against DPPH free radicals was demonstrated.

[0045] Figure 20 This shows the effect of AG on LPS-stimulated macrophages. IL-1β , iNOS , TNF-α , IL-6 The impact on gene expression.

[0046] Figure 21 A schematic diagram of AG grafting MSN is shown.

[0047] Figure 22 TEM images of MSN and MSN@AG are shown. Scale bar is 100 nm.

[0048] Figure 23 The DLS particle size distributions of MSN and MSN@AG are shown.

[0049] Figure 24 The zeta potentials for MSN and MSN@AG are shown.

[0050] Figure 25 The FTIR spectra of MSN, AG, and MSN@AG are shown.

[0051] Figure 26 The DPPH scavenging activities of MSN, AG, and MSN@AG physical mixtures at concentrations of 10 μg / mL and 40 μg / mL are shown. ns: No significant difference, * p <0.05,** p <0.01, *** p <0.001, **** p <0.0001.

[0052] Figure 27 A schematic diagram of the fabrication of the bioactive scaffold SPMA is shown.

[0053] Figure 28 SEM images of different nanofiber scaffolds are shown. Scale bar is 1 μm.

[0054] Figure 29 Histograms showing the fiber diameter distribution of different nanofiber scaffolds are presented.

[0055] Figure 30 The distribution of MSN@AG in SPMA fibers is shown.

[0056] Figure 31 Macroscopic photographs and images of hemolysis experiments of different nanofiber scaffolds are shown.

[0057] Figure 32 The results of energy dispersive X-ray spectroscopy (EDS) elemental analysis of different nanofiber scaffolds are shown.

[0058] Figure 33 The water contact angle measurements of different nanofiber scaffolds are shown.

[0059] Figure 34 The time-series DPPH radical scavenging activity of different nanofiber scaffolds is shown.

[0060] Figure 35 Representative photographs of ABTS (A) and DPPH (B) radical scavenging experiments on different nanofiber scaffolds are shown.

[0061] Figure 36 Quantitative results of ABTS (A), DPPH (B), and fructosamine (C) scavenging rates of different nanofiber scaffolds are shown.

[0062] Figure 37 The results of NO release from LPS-stimulated RAW264.7 macrophages as measured by the Griess method are shown.

[0063] Figure 38 This demonstrates the effects of different nanofiber scaffolds on LPS-stimulated macrophages. IL-6 (A) CD86 (B) TNF-α (C) iNOS (D) Effects on gene expression.

[0064] Figure 39 Immunostaining results of RAW264.7 macrophages under different treatment conditions are shown. iNOS (green), F-actin (red), and DAPI (blue). Scale bar is 20 μm. ns: no significant difference, * p <0.05,** p <0.01, *** p <0.001, **** p <0.0001.

[0065] Figure 40 Representative images of HUVECs formed in Matrigel tubes under different treatment conditions are shown. Scale bar is 500 μm.

[0066] Figure 41 The quantitative analysis of the total tube length (A) and number of nodes (B) of HUVECs in Matrigel tube formation experiments under different treatment conditions is shown.

[0067] Figure 42 HUVECs under different treatment conditions are shown VEGF (A) and CD31 (B) Gene expression status.

[0068] Figure 43 The scratch healing results of MSCs under different treatment conditions are shown (0 h, 24 h, 48 h). Scale bar is 50 μm.

[0069] Figure 44 The results of quantitative analysis of the healing rate of MSCs at 24 h (A) and 48 h (B) under different treatment conditions are shown. p <0.01, *** p <0.001, **** p <0.0001.

[0070] Figure 45 The proliferation curves of CCK-8 cells under different treatment conditions are shown (1 day, 3 days, 5 days).

[0071] Figure 46 Live-cell fluorescence imaging (calcein-AM, green) under different treatment conditions is shown.

[0072] Figure 47 Osteogenic marker genes under different treatment conditions are shown. RUNX2 (A) OPN (B) OCN Gene expression status of (C). p <0.05,** p <0.01, *** p <0.001, **** p <0.0001. Data are expressed as mean ± SD.

[0073] Figure 48 The results of ALP staining (A) on day 7 and ARS staining (B) on day 21 under different treatment conditions are shown.

[0074] Figure 49 Representative 3D Micro-CT reconstructed images are shown at 6 and 12 weeks after implantation of bioactive scaffolds under different treatment conditions.

[0075] Figure 50 Quantitative micro-CT morphological analysis of BV / TV (A), Tb.Th (B), Tb.Sp (C), and Tb.N (D) at 6 weeks and at 12 weeks under different treatment conditions is shown. ns: No significant difference, * p <0.05,** p <0.01, *** p <0.001, **** p <0.0001.

[0076] Figure 51 HE staining results (A) and Masson trichrome staining results (B) of sagittal sections at 12 weeks under different treatment conditions are shown.

[0077] Figure 52 Representative immunofluorescence staining results of OCN(A) and CD31(B) in regenerated tissues under different treatment conditions are shown. The scale bar of the overall image is 200 μm, and the scale bar of the magnified local image is 25 μm. Detailed Implementation

[0078] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0079] Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0080] Experimental methods : 1. Extraction of SSAD from the skin secretions of the giant salamander Data was collected using a non-invasive stimulation method. The giant salamanders were briefly and gently massaged to induce mucus secretion. The collected coarse mucus was immediately diluted with deionized water and stirred at 4°C for 24 hours. Subsequently, the mixture was centrifuged at 10,000 rpm, 4°C for 20 minutes to remove cell debris and impurities. After collecting the supernatant, it was dialyzed against deionized water (MWCO: 3.5 kDa) for 48 hours to remove salts and small molecules. Finally, the purified SSAD was lyophilized to obtain a lyophilized powder of giant salamander skin secretions, which was stored at -80°C for subsequent use.

[0081] 2. Preparation of SPMA bioactive membranes To achieve sustained drug delivery and impart intrinsic anti-inflammatory and antioxidant properties, this invention incorporates SSAD-loaded MSN@AG into a PCL matrix and prepares an SPMA composite scaffold via electrospinning. A schematic diagram of the process is shown below. Figure 27 As shown. The specific steps are as follows:

[0082] 10 g of PCL and 2.5 g of SSAD were dissolved in 10 mL of hexafluoroisopropanol (HFIP) and stirred for 24 h to obtain a mixed solution. Then, 62.5 mg of pre-prepared AG-loaded MSN (MSN@AG) was added to the mixed solution, and stirring was continued for another 24 h to obtain the final spinning solution. All solutions were stirred overnight until a clear and homogeneous solution was obtained.

[0083] Draw up the prepared spinning solution using a 10 mL disposable plastic syringe. After drawing up, hold the syringe vertically upward and slowly push the plunger to completely expel any remaining air from the syringe cavity and needle tip. Then, install a 19 G inner diameter stainless steel blunt-tipped needle at the tip of the syringe. Securely mount the filled syringe onto a precision micro-injection pump and adjust the pump parameters to control the solution to flow outward at a constant rate of 0.0013 m / s.

[0084] Equipment coordination and electrospinning parameter control: This experiment employed a coordinated electrospinning system consisting of a micro-injection pump, a high-voltage power supply, and a roller collector. Spatial collaborative architecture: A syringe with a stainless steel needle was horizontally fixed, its tip facing a rotating roller collector covered with highly conductive aluminum foil. The vertical receiving distance between the needle tip and the aluminum foil surface was precisely adjusted to 15 cm. Electric field and collection coordination: The positive output of the high-voltage DC power supply was securely clamped to the stainless steel needle using alligator clips, while the negative terminal was safely connected to the bearing of the roller collector, thus establishing a uniform high-voltage electrostatic field between the needle tip and the aluminum foil roller. Operating parameter settings: The system was started, and the applied voltage was adjusted to 18 kV. Simultaneously, the roller collector was started, maintaining its rotation speed at 800 rpm. Under stable electrostatic repulsion, the droplet at the needle tip formed a stable Taylor cone and ejected as a jet. After solvent evaporation in space, nanofibers were uniformly deposited on the rotating aluminum foil surface. The entire spinning process is carried out in a temperature and humidity controlled environment (temperature: 25±2°C, relative humidity: 40%±5%), with continuous spinning for 10 hours to obtain a film of the required thickness (0.5 mm).

[0085] After spinning, the high-voltage power supply is turned off and a discharge process is performed. The aluminum foil with the nanofiber membrane is carefully removed from the roller. To completely remove any trace organic solvents remaining inside the fibers, the holder with the aluminum foil is placed in a vacuum drying oven and vacuum dried at 37°C for 48 hours. Finally, the completely dried nanofiber membrane is gently peeled off the aluminum foil, cut to the specific size required for the experiment using a punch or scissors, and sterilized for later use.

[0086] 3. Preparation method of MSN@AG nanoparticles Specifically, 80 mg of MSN was dispersed in 10 mL of absolute ethanol containing 400 mg of AG (AG to MSN mass ratio 5:1). The suspension was sonicated for 15 minutes and then stirred at room temperature for 24 hours to allow AG to permeate the mesopores. The mixture was then centrifuged and the MSN@AG was washed repeatedly with ethanol to remove surface-adsorbed AG.

[0087] 4. Experimental grouping and component dosage Group P: 10 g PCL + 10 mL hexafluoroisopropanol; SP group: 10 g PCL + 10 mL hexafluoroisopropanol + 2.5 g SSAD; SPM group: 10 g PCL + 10 mL hexafluoroisopropanol + 2.5 g SSAD + 62.5 mg MSN; SPMA group: 10 g PCL + 10 mL hexafluoroisopropanol + 2.5 g SSAD + 62.5 mg MSN@AG; SPA group: 10 g PCL + 10 mL hexafluoroisopropanol + 2.5 g SSAD + 62.5 mg AG.

[0088] 5. BMSCs cell scratch migration assay (1) Cell seeding: Seed cells in 6-well plates and culture until 100% complete confluence; (2) Serum starvation: starve the medium with low serum (0.5%, v / v) or serum-free medium for 12 hours; (3) Precise scratching: Use a 200 µL sterile pipette tip to make vertical scratches, and gently wash with warm PBS 2-3 times to thoroughly remove floating cell debris; (4) Drug addition culture: Replace with 2 mL serum-free culture medium containing 400 µL of SP and SPMA extracts from each group and continue culture.

[0089] (5) Fixed-point photography: Take photos at 0 hours, 12 hours and 24 hours respectively, under the same field of view through the bottom mark of the board.

[0090] (6) Quantitative analysis: The area of ​​the scratch was measured using ImageJ software.

[0091] 6. Preparation of SP extract and SPMA extract SP or SPMA scaffolds were immersed in whole-cell medium (α-MEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin) or Dulbecco modified Eagle medium (DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin) at a scaffold-to-medium volume ratio of 0.2 g / mL. The mixture was incubated at 37°C for 24 hours with continuous shaking (80 rpm), filtered through a 0.22 µm sterile filter, and stored at 4°C until use (within 1 week). The pH of all extracts was confirmed to be 7.2–7.4 prior to the experiment.

[0092] 7. Gene detection after LPS stimulation of macrophages (1) Take 0.5 µL of LPS stock solution with a concentration of 1 mg / mL and place it in 2 mL of RAW264.7 macrophage culture medium (Visent, 319-005-CL). After culturing for 24 h, replace it with serum-free culture medium containing 400 µL of SP and SPMA extracts from each group and continue culturing for 24 h.

[0093] (2) RNA was extracted from cells using an RNA kit (RN001, Shanghai Yishan).

[0094] (3) Reverse transcription was performed using a reverse transcription kit (R312, Vazyme).

[0095] (4) Quantitative analysis using qpcr kit (Q712, Vazyme).

[0096] 8. BMSCs scratch healing experiment BMSCs were seeded in 6-well plates. Once cell confluence reached 100%, vertical streaks were made on the bottom of each well using a 200 μL sterile pipette tip. After washing with PBS to remove suspended cells, the culture medium was replaced with the extraction medium from each scaffold group: 2 mL of DMEM low-glucose medium (PronoYi Biotechnology, AM1004) with 400 μL of SP / SPMA extraction medium. The cells were then cultured at fixed time points (0 h, 12 h, 24 h) using an inverted microscope and photographed. The scratch closure rate was quantitatively calculated using ImageJ software. The high-glucose (HG) group consisted of 2 mL of DMEM low-glucose medium with 12.6 mg of glucose added.

[0097] Example 1: Preparation and Characterization of SP Bioactive Membranes 1.1 Preparation of SP bioactive membranes The preparation process of SP bioactive membrane is as follows: Figure 1 As shown.

[0098] SSAD was successfully extracted from the skin secretions of the giant salamander by freeze-drying. Subsequently, it was added to PCL solution at different weight ratios (PCL:SSAD = 10:1, 4:1, 2:1, w / w) for electrospinning. Figure 2 As shown.

[0099] The electrospinning process is the same as that used in SPMA preparation.

[0100] 1.2 Characterization of SP bioactive membranes The results showed that the obtained electrospun film was flexible, uniform, and easy to peel off from the collector. The macroscopic appearance became slightly less transparent with increasing SSAD loading. Figure 3 Scanning electron microscopy (SEM) revealed that all scaffolds exhibited highly porous, bead-free, and randomly oriented nanofiber structures, consistent with typical PCL electrospinning characteristics. Figure 4 The fiber diameters of each group were similar, mainly distributed in the range of 300 nm to 800 nm. There was no statistically significant difference in the average diameter and distribution between the pure PCL and SSAD-loaded groups. Figure 5 FTIR spectroscopy confirmed successful SSAD doping: pure PCL at 2945 cm⁻¹ -1 (CH stretching) and 1160 cm -1 Characteristic peaks are present at the (COC expansion) location; the SSAD load-bearing support retains these peaks at 1650 cm⁻¹. -1 A broadband band of amide I / II appears at 2800–3000 cm⁻¹ (a characteristic of SSAD protein / peptide). -1Regional intensity increased, with the most significant change observed in a 2:1 ratio. Figure 6 ).

[0101] Results of in vitro release experiments are as follows Figure 7 As shown, SSAD was released continuously from the stent in a load-dependent manner over 14 days. Pure PCL showed almost no release, while the SSAD-loaded group showed an initial burst followed by continuous release. The 2:1 ratio showed the highest cumulative release and the fastest initial rate, while the 10:1 and 4:1 ratios provided more controllable release profiles, suitable for long-term bioactivity.

[0102] The water contact angle measurement results of the support surface are as follows: Figure 8 As shown, surface wettability significantly improves with dose-dependent SSAD incorporation. The contact angle of pure PCL is approximately 140° (hydrophobic), which gradually decreases with increasing SSAD content, reaching the lowest value in the 2:1 group (60°–70°), indicating a significant enhancement in hydrophilicity.

[0103] Example 2: Biocompatibility and osteogenic induction capacity of SP To determine the optimal SSAD loading concentration, live-cell fluorescence imaging (calcein-AM, green) was performed on scaffolds with different PCL:SSAD mass ratios (pure PCL, 10:1, 4:1, 2:1 w / w) at 1 and 3 days. Results showed that in the pure PCL group, cell adhesion was sparse at 1 day and proliferation was limited at 3 days; while in the SSAD-loaded group, cell adhesion and spreading were significantly enhanced in a dose-dependent manner, with the 4:1 and 2:1 ratios approaching monolayer fusion at 3 days, demonstrating excellent cell compatibility and proliferation capacity. Figure 9 Therefore, a PCL:SSAD mass ratio of 4:1 was selected as the optimal concentration for subsequent formal experiments.

[0104] After co-culturing HUVECs with SSAD extract, the formation of capillary-like networks was significantly enhanced. Figure 10 qRT-PCR confirmed that CD31 and VEGF expression was upregulated in the SSAD treatment group. Figure 11 The CCK-8 assay showed that cell viability gradually increased in all groups from day 1 to day 5, with the optimal concentration of SSAD loading showing significantly higher absorbance than the PCL group at days 3 and 5. Figure 12 Live and dead cell staining further confirmed excellent cell compatibility; the SSAD group showed >95% live cells (green) and very few dead cells (red). Figure 13 ).

[0105] Osteogenic potential was assessed by ALP staining on day 7 and ARS staining on day 21. Figure 14The SSAD-loaded group showed significantly enhanced ALP activity, with significantly more calcium nodule formation and matrix mineralization compared to the PCL group. qRT-PCR results consistently showed that the osteogenic marker genes OCN, OPN, and RUNX2 were significantly upregulated in the SSAD group. Figure 15 ).

[0106] Example 3 Characterization of MSN@AG nanoparticles The results showed that free AG exhibited concentration-dependent antioxidant and anti-inflammatory effects in vitro. CCK-8 (Solarbio, CA1210) assays showed that AG (0 μM–100 μM) had no significant cytotoxicity on RAW264.7 macrophages, with only a slight decrease in viability at high concentrations. Figure 16 AG significantly inhibited LPS-induced NO release in a dose-dependent manner, reducing NO release (Beyotime, S0020) to near baseline levels at ≥50 μM. Figure 17 In cell-free free radical scavenging experiments, AG showed strong scavenging activity against both ABTS (Beyotime, S0121) and DPPH (Beyotime, A003) free radicals, with significantly increased scavenging rates in the range of 20 nM to 400 nM. Figure 18 and Figure 19 qRT-PCR results showed that AG (10 μM) significantly downregulated LPS-induced cytokinesia. IL-1β , iNOS , TNF-α and IL-6 The expression of pro-inflammatory factors and M1 marker genes is reduced by 50% to 80% or more. Figure 20 ).

[0107] Successful grafting of AG onto the MSN surface (MSN@AG) was confirmed by TEM (maintaining a ~100 nm spherical morphology with slight aggregation), DLS (similar particle size distribution), Zeta potential (shift from negative to positive potential, indicating successful cation modification), and FTIR (appearance of AG characteristic peaks). Figures 21-25 MSN@AG maintained strong DPPH scavenging activity at both 10 μg / mL and 40 μg / mL (Beyotime, A003), comparable to or slightly higher than free AG, demonstrating that the antioxidant function was preserved after grafting. Figure 26 The above results indicate that free AG possesses inherent strong antioxidant and anti-inflammatory properties, providing mechanistic support for the enhanced bioactivity of MSN@AG in SPMA scaffolds.

[0108] Example 4 Characterization of SPMA bioactive membrane SEM analysis showed that PCL, SP, SPM, and SPMA scaffolds with different SSAD:MSN@AG mass ratios (20:1, 40:1, 100:1 w / w) all exhibited highly porous, bead-free, and interconnected nanofiber structures. Figure 28 The incorporation of nanoparticles slightly roughened the fiber surface but did not disrupt the structural integrity. The fiber diameters in each group were similar, mainly distributed between 300 nm and 800 nm, with no significant differences. Figure 29 Fluorescence microscopy confirmed that MSN@AG was uniformly dispersed in SPMA fibers, and the fluorescence intensity increased with increasing nanoparticle loading (strongest at a ratio of 20:1). Figure 30 ).

[0109] Macroscopic images show that the SPMA stent is flexible and easy to manipulate, with potential for minimally invasive applications. Figure 31 Energy-dispersive X-ray spectroscopy (EDS) elemental analysis confirmed that MSN@AG nanoparticles were successfully and uniformly incorporated into the fiber, and that Si (characteristic of silicon cores) was uniformly distributed within the SPMA scaffold. Figure 32 The surface wettability of the nanoparticle-containing scaffold was significantly improved, with the water contact angle gradually decreasing from SP to SPM and then to SPMA, with SPMA showing the lowest angle. Figure 33 This indicates that AG functionalized nanoparticles significantly enhance hydrophilicity.

[0110] The FTIR transmission spectra of P, SP, SPM, and SPMA scaffolds retained the characteristic peaks of PCL (~2945-2865 cm⁻¹). -1 For CH stretching, ~1720 cm -1 For ester C=O, 1240 cm -1 ~1160 cm -1 For COC). SPMA and SPM at 1650 cm -1 ~1550 cm -1 A weak peak for amide I / II appeared in the region, at 3000 cm⁻¹ -1 ~2800 cm -1 Enhanced regional strength confirms successful and uniform incorporation of MSN@AG nanoparticles without causing chemical changes or degradation of the PCL matrix. The DPPH radical scavenging activity of the scaffold extracts changed over time (5 min to 168 h): the SPA extract showed a rapid decline after an initial burst release, while the SPMA extract exhibited more stable and sustained scavenging activity, maintaining a high scavenging rate throughout the observation period, indicating that MSN@AG encapsulation prolonged the antioxidant effect. Figure 34 ).

[0111] Example 5: Determination of the anti-inflammatory and free radical scavenging capabilities of SPMA Antioxidant capacity was assessed using free radical scavenging assays with ABTS (Beyotime, S0121), DPPH (Beyotime, A003), and fructosamine (Beyotime, ST1222). The photographs show significant decolorization in the nanoparticle-containing groups (SPM and SPMA), with SPMA showing the most significant effect. Figure 35 Quantitative analysis confirmed that the SPMA extract had the highest clearance rate in all three experiments, significantly better than the P and SP groups. Figure 36 This indicates that MSN@AG incorporation endows the organism with excellent broad-spectrum free radical scavenging activity.

[0112] 0.5 µL of a 1 mg / ml LPS stock solution was added to 2 mL of RAW264.7 macrophage culture medium. 400 µL of SP extract and SPMA extract were then added to the medium. Griess method showed that LPS significantly increased NO release, which was significantly inhibited by SP extract, with SPMA extract showing the strongest inhibitory effect. Figure 37 qRT-PCR results showed that LPS was strongly upregulated. IL-6 , CD86 , TNF-α and iNOS The expression of pro-inflammatory factors and M1 marker genes was reduced; both SP extract and SPMA extract could downregulate these genes, with SPMA showing the most significant inhibitory effect, often reducing the expression levels to near or below those of the NC group. Figure 38 Immunofluorescence staining results were consistent: LPS stimulation significantly enhanced iNOS expression (green), and F-actin polymerization (red) was obvious, with cells exhibiting a typical M1-like spreading morphology; SP extract partially alleviated iNOS and cytoskeleton remodeling, while SPMA extract significantly reduced iNOS fluorescence intensity, restored dense F-actin structure, and cell morphology was close to that of the unstimulated NC group. Figure 39 ).

[0113] In summary, the SPMA scaffold possesses strong antioxidant activity and the ability to inhibit macrophage M1 polarization, effectively regulating the inflammatory and oxidative microenvironment in diabetic bone repair.

[0114] Example 6: SPMA can promote tube formation and recruit stem cells The angiogenesis-promoting capacity of SPMA scaffolds was assessed using HUVECs. A Matrigel tube formation assay (Beyotime, C0372) showed that capillary-like network formation in the SPMA group was significantly superior to that in the high glucose control (HG) and SP groups. Figure 40 The SPMA group exhibits longer, more branched, and better-connected tubular structures. Quantitative analysis shows a significant increase in the total tubular length and number of nodes in the SPMA group. Figure 41Consistent with the phenotype, SPMA significantly upregulated the mRNA expression of key angiogenesis marker genes such as VEGF and CD31. Figure 42 ).

[0115] To evaluate the effect on bone regeneration-related cell migration, BMSCs were used in a scratch healing assay. Time-series imaging showed that the SPMA extract treatment group exhibited significantly faster scratch healing, reaching near-complete fusion earlier than the HG or SP groups. Figure 43 Quantitative results of migration area and migration rate confirmed that the SPMA group had the strongest migration-promoting effect. Figure 44 ).

[0116] The above results indicate that the SPMA composite scaffold can significantly promote endothelial cell tube formation and angiogenesis gene expression under high glucose conditions, while enhancing the migration ability of BMSCs, providing strong support for vascularized bone regeneration in the diabetic microenvironment.

[0117] Example 7: SPMA Biocompatibility and In Vitro Osteogenic Potential Determination To evaluate the biocompatibility and osteogenic induction capacity of SPMA scaffolds under diabetic-like conditions, MSCs were cultured under the following five conditions: ambient temperature 37°C, carbon dioxide concentration 5%. Cells were passaged using a cell scraper at 70%–80% confluence and used at passages 5–10. The experimental groups are shown below:

[0118] Normal control group (NC): 50 mL low-glucose culture medium (Pronobio, AM1004). Low-glucose osteogenic induction medium group (Oi, low-glucose + osteogenic induction): 50 mL osteogenic induction medium (OriCell, RAXMX-90021) + 50 mg glucose; High glucose osteogenic induction medium group (HOi, high glucose + osteogenic induction): 50 mL osteogenic induction medium (OriCell, RAXMX-90021) + 315 mg glucose; SP extract treatment group under high glucose conditions (SP-HG): 20 mL SP scaffold extract + 30 mL osteogenic induction medium (OriCell, RAXMX-90021) + 315 mg glucose; SPMA extract treatment group under high glucose conditions (SPMA-HG): 20 mL SPMA scaffold extract + 30 mL osteogenic induction medium + 315 mg glucose.

[0119] The CCK-8 assay showed that all groups of cells exhibited a good proliferation trend within 5 days. Figure 45Among them, the SPMA-HG group showed the highest cell viability at all time points, reflecting the optimal osteogenic microenvironment. Notably, the proliferation capacity of the SPMA-HG group was significantly higher than that of the HG-P, HG-SP, and HG-SPM groups.

[0120] Fluorescence live-cell imaging further confirmed the enhanced cell adhesion and fusion: the Oi group (low-glucose osteogenic induction) had the densest monolayer on day 3, followed by the SPMA-HG group, while the HOi and SP-HG groups had significantly lower cell densities. Figure 46 qRT-PCR analysis showed that the expression of osteogenic marker genes (RUNX2, OPN, OCN) was significantly upregulated in the SPMA-HG group. Figure 47 The results of ALP staining (Beyotime, P0321M) (day 7) and ARS staining (OriCell, ALIR-10001) (day 21) consistently showed that the SPMA-HG group had the strongest alkaline phosphatase activity, the most calcium nodules, and the highest degree of mineralization. Figure 48 ).

[0121] Overall, the low-glucose Oi group showed the best osteogenic induction effect, followed by the SPMA-HG group, which was significantly better than the high-glucose HOi group and the SP-HG group. This indicates that the SPMA scaffold can effectively reverse osteogenic inhibition in the high-glucose diabetic microenvironment and restore osteogenic differentiation and mineralization capacity to near-normal levels.

[0122] Example 8 SPMA in vivo osteogenic potential determination Procedure for constructing a diabetic healing disorder mouse model: First, 8-week-old C57 / 6J mice (approximately 15-20 g) were selected and fasted for 12 hours before induction, but allowed free access to water. Simultaneously, 120 mg of streptozotocin (STZ) was accurately weighed and dissolved in 8 mL of pre-cooled, freshly prepared 0.1 mol / L sodium citrate buffer (pH 4.5). The solution was quickly stirred thoroughly in the dark and used within 30 minutes to prevent STZ degradation. Next, a single high-dose intraperitoneal injection of 150 mg / kg was administered, proportional to the mouse's body weight. Following injection, because STZ damages pancreatic β-cells, causing a transient surge in insulin release, to prevent immediate death from fatal hypoglycemia, a 5% glucose solution was provided in the mice's drinking water for the first 24 hours after injection. Afterward, normal drinking water and regular feeding were resumed. On days 3 and 7 after induction, fasting blood glucose levels were measured using a precision blood glucose meter (Yuwell, China) via tail vein puncture. A diabetic mouse model is considered successfully established when the fasting blood glucose level of mice is greater than or equal to 16.7 mmol / L (or 300 mg / dL) in two consecutive measurements, and the mice exhibit typical diabetic symptoms such as polydipsia, polyphagia, polyuria, and weight loss. This model can then be used for subsequent scaffold implantation and bone repair evaluation experiments.

[0123] This invention provides a method for constructing a mouse animal model and implanting a scaffold. Specifically, general anesthesia is administered using isoflurane gas inhalation. Once the mouse is deeply anesthetized and shows no spontaneous reflex to needle prick, it is fixed to a sterile operating table. The area to be operated on in the mouse skull is shaved using electric shaving shears, followed by three alternating wipes with povidone-iodine from the inside out for disinfection, and then a sterile surgical drape is laid. Next, the skin and subcutaneous tissue are incised in the disinfected area using a sterile scalpel, and a bone defect with a diameter of 3 mm is created using a bone drill to prepare the implantation space required for the experiment. During the scaffold preparation and implantation stage, a pre-prepared composite bioactive scaffold membrane is precisely cut into 1.5 cm × 3 cm rectangular films using precision surgical scissors under sterile conditions, and then thoroughly sterilized using cobalt-60 irradiation. Subsequently, under aseptic conditions, using two ophthalmic forceps, the 1.5 cm × 3 cm bioactive scaffold membrane was folded to reduce its area and increase its local thickness. The folded scaffold was then carefully placed into the surgical incision defect of the mouse, its position adjusted to ensure complete containment and close adhesion to surrounding tissue. After implantation, the gap and external skin incision were meticulously sutured with sterile medical sutures, and an appropriate amount of antibiotic ointment was applied to the wound surface to prevent postoperative infection. Finally, the mice were placed on a 37°C heated blanket for rewarming. Once fully awake and able to move freely, they were transferred back to the animal room for routine cage rearing. Postoperatively, the mice's diet, wound healing, and overall condition were regularly observed.

[0124] Subsequent experiments were conducted in NC (normal mice), P, SP, SPM and SPMA groups, with 5 replicates in each group.

[0125] This invention provides a method for Micro-CT scanning and three-dimensional reconstruction analysis of bone defects in mice. Specifically, mice are sacrificed at 6 and 12 weeks of age, and tissue samples containing the implanted scaffold from the skull defect site are harvested intact. These samples are immediately immersed in a 4% polyoxymethylene solution for at least 48 hours for fixation. Subsequently, the fixed bone specimens are removed, washed with sterile saline, and blotted dry with filter paper. They are then securely fixed on the sample testing stage of the Micro-CT system for high-resolution X-ray scanning. During the scanning process, the core physical parameters of the system are set as follows: source voltage 55 kV, source current 180 μA, 0.5 mm aluminum filter, rotation step size set to 0.4°–0.6°, and spatial resolution (voxel size) precisely controlled between 9 μm and 15 μm. A series of high-resolution two-dimensional projection raw images are obtained by continuous rotation scanning for one revolution. After scanning, the original two-dimensional projection data was reconstructed into three-dimensional tomographic images using the system's accompanying tomographic reconstruction software, NRecon. During reconstruction, precise beam hardening correction, ring artifact elimination, and smoothness optimization were performed by adjusting algorithm parameters to obtain continuous cross-sectional tomographic pixel images. Next, the reconstructed tomographic images were uniformly imported into the three-dimensional data analysis software CTAn. Using the original bone defect area (i.e., the central area of ​​the scaffold implantation) as the geometric core, specific layers were extended outwards and vertically to strictly define a standardized stereoscopic region of interest (ROI). After determining the ROI, precise digital segmentation of the newly formed bone tissue from the surrounding low-density soft tissue, fat, or residual scaffold material was performed by setting a fixed grayscale threshold. Finally, high-resolution 3D morphological reconstruction images of newly formed bone in the bone defect area are generated using 3D visualization rendering software, such as CTVox or Inveon, to comprehensively demonstrate the healing status of the bone defect. At the same time, key bone morphological parameters, including bone mass fraction (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp), are automatically calculated and output through a quantitative analysis module to objectively and quantitatively evaluate the repair effects of different groups (P, SP, SPM, SPMA) scaffolds on promoting in vivo bone regeneration in the diabetic pathological microenvironment.

[0126] Micro-CT 3D reconstruction (Hiscan XM, China) shows that new bone formation in the defect area progresses gradually over time. Figure 49 At 6 weeks, regeneration was limited in all implantation groups, with the SPMA group showing the most significant radiopermeability bridging. At 12 weeks, large areas of defects remained unfilled in the P and SP groups, the SPM group showed moderate improvement, while the SPMA-treated group showed near-complete defect closure with extensive mineralized tissue filling, and the structure was close to that of the intact NC group. Quantitative morphological analysis further confirmed this result. Figure 50At 6 and 12 weeks, the SPMA group showed the best bone volume fraction, trabecular thickness, trabecular separation, trabecular number, and bone mineral density among all implantation groups, significantly superior to the P, SP, and SPM groups. At 12 weeks, it surpassed the non-diabetic NC group, indicating that SPMA effectively reversed diabetic healing impairment. Figure 50 ).

[0127] Histological evaluation was used to assess the quality and maturity of regenerated bone in diabetic cranial defects. HE staining showed the progression of tissue remodeling over time. At 6 weeks, the defects in all implantation groups were mainly filled with loose fibrous connective tissue, with limited marginal new bone formation. Early osteogenic formation was most pronounced in the SPMA group, with increased osteoid matrix and osteoblast arrangement. At 12 weeks, the P and SP groups were still dominated by fibrous tissue with incomplete bone bridging, while the SPM group showed moderate improvement. The SPMA-treated group showed extensive mature bone regeneration, characterized by thick lamellar trabeculae crossing the defect and integrating with the host bone, with histological morphology approaching that of the intact NC group. Figure 51 Masson trichrome staining showed enhanced collagen deposition and maturation: at 6 weeks, collagen fibers (blue) were sparse and disordered in all groups; at 12 weeks, the SPMA group showed a significant increase in collagen content and tissue formation, with dense and well-arranged fiber bundles indicating advanced matrix remodeling and bone maturation, significantly superior to the P, SP, and SPM groups. Figure 51 Immunofluorescence analysis () Figure 52 Quantitative statistical analysis confirmed significantly enhanced expression of osteogenic protein (OCN) and angiogenesis (CD31) in regenerated tissue. The SPMA group showed the strongest staining of key markers, with signals concentrated in newly formed bone and related blood vessels, further supporting accelerated and high-quality bone regeneration under diabetic conditions. These histological results are highly consistent with Micro-CT data, indicating that the SPMA scaffold can promote mature bone formation, collagen maturation, and bioactive protein expression, effectively overcoming diabetic obstacles and achieving regenerative effects close to those of the NC group.

[0128] Twelve weeks after implantation, representative HE-stained sections of major organs (heart, liver, spleen, lung, and kidney) were collected from the NC, P, SP, SPM, and SPMA groups. No pathological changes, inflammatory cell infiltration, fibrosis, necrosis, or stent-related toxicity were observed at any time point in any group, confirming that SPMA has excellent long-term biocompatibility and no systemic adverse reactions.

[0129] It should be noted that, since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of the present invention have been described, those skilled in the art, once they understand the inventive concept of the present invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of the present invention.

[0130] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention also intends to include these modifications and variations.

Claims

1. A bioactive scaffold, characterized in that, The bioactive scaffold was obtained by integrating giant salamander skin secretions and mesoporous silica nanoparticles loaded with aminoguanidine into polycaprolactone; and the bioactive scaffold has a nanofiber structure. Among them, the skin secretions of the giant salamander are loaded in mesoporous silica nanoparticles containing aminoguanidine; The mesoporous silica nanoparticles loaded with aminoguanidine are obtained by grafting aminoguanidine onto the surface of mesoporous silica nanoparticles. The mass ratio of the aminoguanidine to the mesoporous silica nanoparticles is 5:

1.

2. The bioactive scaffold according to claim 1, characterized in that, The diameter of the nanofiber structure is 300 nm to 800 nm.

3. The bioactive scaffold according to claim 2, characterized in that, The giant salamander skin secretion is a freeze-dried powder of giant salamander skin secretion, and the mass ratio of polycaprolactone to freeze-dried powder of giant salamander skin secretion is 4:

1.

4. The bioactive scaffold according to claim 3, characterized in that, The mass ratio of the freeze-dried powder of the giant salamander skin secretions to the mesoporous silica nanoparticles loaded with aminoguanidine is 20:1 to 100:

1.

5. The bioactive scaffold according to claim 4, characterized in that, The mass ratio of the freeze-dried powder of the giant salamander skin secretions to the mesoporous silica nanoparticles loaded with aminoguanidine is 40:

1.

6. The method for preparing a bioactive scaffold according to any one of claims 1 to 5, characterized in that, The preparation method includes: (1) Dissolve polycaprolactone and freeze-dried powder of giant salamander skin secretions in hexafluoroisopropanol, mix well to obtain a mixed solution; (2) Add mesoporous silica nanoparticles loaded with aminoguanidine to the mixed solution and mix them evenly to obtain a spinning solution; (3) The spinning solution is electrospun to obtain the bioactive scaffold; The mass-to-volume ratio of polycaprolactone to hexafluoroisopropanol is 1:1; The mass-to-volume ratio of the freeze-dried powder of the giant salamander skin secretions to the hexafluoroisopropanol is 1:

4.

7. The preparation method according to claim 6, characterized in that, The method for preparing the mesoporous silica nanoparticles loaded with aminoguanidine includes: Mesoporous silica nanoparticles were dispersed in anhydrous ethanol containing aminoguanidine, and then ultrasonically treated to mix them evenly. After centrifugation and washing, mesoporous silica nanoparticles loaded with aminoguanidine were obtained. The mass-to-volume ratio of the aminoguanidine to the absolute ethanol is 1:

25.

8. The preparation method according to claim 7, characterized in that, The electrospinning conditions were: temperature 25±2°C, relative humidity 40%±5%, and voltage parameter set to 18 kV. The spinning solution is propelled through a needle with an inner diameter of 19G at a constant flow rate of 0.0013 m / s into the roller collector of the electrospinning system. The roller collector rotates at 800 rpm, and the vertical receiving distance from the needle tip to the surface of the roller collector is set to 15 cm.

9. Use of the bioactive scaffold according to any one of claims 1 to 5 in the preparation of a medicament for repairing diabetic bone defects.

10. The use according to claim 9, characterized in that, The drug is used to scavenge free radicals and / or regulate inflammation; and / or The drug is used to promote angiogenesis and / or bone regeneration-related cell migration.

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