Asymmetric short fiber aerogel stent loaded with magnesium oxide as well as preparation method and application of asymmetric short fiber aerogel stent

Abstract

The invention provides a magnesium oxide-loaded asymmetric short fiber aerogel scaffold as well as a preparation method and application thereof, and belongs to the technical field of tissue engineering materials. The preparation method comprises the following steps: adding MgO nanoparticles into a PLLA / Gelatin solution, and carrying out electrostatic spinning, crushing, freeze drying and crosslinking to obtain the porous short fiber aerogel scaffold (GP-coated MgO). The GP-coated MgO aerogel stent has a three-dimensional short fiber structure, and can release loaded MgO nanoparticles in a progressive and continuous manner while wound exudate is slowly absorbed. In-vivo and in-vitro experiments prove that the GP-coated MgO aerogel scaffold has biocompatibility and is very effective in the aspects of inhibiting inflammatory response and slowing down cell senescence. In addition, the GP-coated MgO significantly accelerates vascular reconstruction and epithelization, and promotes wound repair at the skin defect position of a diabetic rat.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue engineering materials and relates to a magnesium oxide-loaded asymmetric short fiber aerogel scaffold and a preparation method and application thereof. Background Art

[0002] As the body's first line of defense, the skin is susceptible to external trauma and internal diseases. With the increasing incidence of diabetes, effective wound management has become increasingly urgent, especially since the incidence of diabetic wounds caused by elevated blood sugar levels and persistent chronic inflammation is also increasing. The persistent disorder of the immune microenvironment in the diabetic state is a major obstacle to the optimal effect of wound repair. Incomplete resolution or dysregulation of acute inflammation leads to the persistence of the local inflammatory microenvironment, causing immune-induced aging of the surrounding tissues. The increased oxidative stress caused by the high-sugar microenvironment can cause damage to the body. This further exacerbates local inflammation, prolongs the inflammatory phase of wound healing, and accelerates inflammatory aging of the host tissue. Therefore, in order to promote the regeneration of skin wounds in the complex microenvironment of diabetes, the use of tissue materials to intervene in the physiological process of wound regeneration has attracted increasing attention.

[0003] The wound healing process is divided into hemostasis, inflammation, proliferation, and remodeling phases. To achieve optimal wound healing, the interplay between the complex diabetic microenvironment and host physiology must be considered to shorten the required time. Innate immunity represents the initial response after injury, driving neutrophil activation and macrophage recruitment. However, in diabetes, the prolonged local presence of M1 macrophages and delayed expression of M2 macrophages are primarily responsible due to high levels of oxidative stress and abnormally active energy metabolic pathways. This macrophage polarization results in wounds stagnating in the inflammatory phase, which can lead to the formation of chronic wounds. Furthermore, due to the influence of multiple external factors, mitochondrial function is impaired, resulting in decreased membrane potential and impaired ATP production. Excessive accumulation of ROS and the persistent secretion of the senescence-associated secretory phenotype (SASP) can lead to premature cellular senescence, creating a vicious cycle. The accumulation of senescent cells also hinders diabetic wound healing.

[0004] Metal ions participate in molecular interactions within the immune system through their structural, catalytic, or regulatory properties and provide a promising foundation for novel metalloimmunotherapies. For example, metal ions can play a role in cell signaling by acting as enzyme cofactors, leading to the activation or enhancement of intrinsic and adaptive immunity. Furthermore, metal ions can influence the expression of inflammatory factors and modulate the composition and growth of the microbiome. This could help alleviate the growing crisis of antibiotic overuse. For example, MgO nanoparticles can competitively inhibit calcium influx into cells by altering the local pH environment, thereby inhibiting cell senescence and restoring cell activity. Consequently, metal nanoparticles have attracted considerable attention in tissue engineering because they are essential trace elements in the human body. However, due to the potential biotoxicity caused by the rapid increase in ion abundance within a short period of time after application, the selection of appropriate delivery systems to control their release rate is crucial, such as decellularized matrices, hydrogels, microspheres, or electrospinning.

[0005] Electrospinning can be used to arrange micro / nanoscale fiber filaments to mimic the structure of natural extracellular matrix. It is crucial to select suitable raw materials to achieve optimal biomimetic performance and provide mechanical support as well as ensure biological survival. Poly (L-lactic acid) (PLLA) is incorporated into it to balance the hydrophilicity of pure gelatin with the inherent lack of mechanical properties of the material. At the same time, electrospinning technology has an extremely high specific surface area, which provides more attachment points for cells, thereby promoting cell adhesion and proliferation, enabling it to play a role in the co-evolution of the cell's intrinsic structure and the precise structural remodeling of the microenvironment.

[0006] However, the traditional method of preparing fiber membranes through electrospinning produces a two-dimensional structure formed by densely stacked fibers with small pores. The lack of a three-dimensional structure poses challenges in practical applications. Furthermore, when attempting to composite metal ions with electrospun fiber membranes, gelatin easily agglomerates with nanoparticles, clogging the spinning nozzle. Furthermore, the fibers are scattered and difficult to collect, resulting in a very small amount of collected fiber membrane. Furthermore, the fiber scaffold is fragile during molding, with disordered fiber stacking and a lack of or limited pore size. This lack of high porosity and three-dimensional structure makes it difficult to ensure rapid cell growth and the exchange of substances and nutrients required for tissue remodeling during wound healing. This results in extremely limited effectiveness in wound repair, particularly for diabetic patients.

[0007] Therefore, how to construct a short fiber aerogel scaffold with high porosity and three-dimensional structure through electrospinning process to achieve the unique morphology and microporous structure of the scaffold, thereby ensuring the exchange of substances and nutrients during cell growth and tissue remodeling, and being well used for wound repair in diabetic patients, has become a technical problem that needs to be solved urgently. Summary of the Invention

[0008] The present invention aims to address the aforementioned technical issues and provides a magnesium oxide-loaded asymmetric short fiber aerogel scaffold, its preparation method, and its application. The technical objectives of the present invention are, on the one hand, to address the problem that existing electrospun fiber scaffolds have insufficient porosity and are mostly two-dimensional or three-dimensional layered disordered structures, making them difficult to support cell growth and tissue remodeling; on the other hand, to provide an aerogel fiber scaffold that can better repair diabetic wounds.

[0009] In order to achieve the above technical objectives, the technical solutions adopted by the present invention are as follows:

[0010] The present invention first provides a method for preparing a magnesium oxide-loaded asymmetric short fiber aerogel scaffold, comprising the following steps:

[0011] (1) Mixing magnesium oxide nanoparticles with a solution of gelatin and L-polylactic acid and stirring for more than 12 hours to prepare an electrospinning solution; then electrospinning the electrospinning solution under the following conditions: voltage 17-19 kV, rotation speed 300 rpm, flow rate 3 mL / h, and collection distance 15 cm to obtain a nanofiber membrane;

[0012] (2) The nanofiber membrane obtained in step (1) is cut into pieces and then prepared into a short fiber suspension. The short fiber suspension is ground at 25 Hz for 10 minutes, then allowed to settle for 15 minutes, freeze-dried, and thermally cross-linked to prepare a short fiber aerogel scaffold.

[0013] The above-mentioned method of the present invention prepares a porous short fiber aerogel scaffold by adding MgO nanoparticles to a PLLA / Gelatin solution and preparing it through electrospinning, crushing, freeze drying and cross-linking. The Gelatin / PLLA@MgO (GP@MgO) aerogel scaffold with a three-dimensional short fiber structure designed by the present invention can release the loaded MgO nanoparticles in a gradual and continuous manner while slowly absorbing wound exudate. In vivo and in vitro experiments have demonstrated that the GP@MgO aerogel scaffold is biocompatible and very effective in inhibiting inflammatory responses and slowing cell aging. In addition, GP@MgO significantly accelerated vascular reconstruction and epithelialization, promoting wound repair in skin defects of diabetic rats.

[0014] During the preparation of the above-mentioned short fiber aerogel scaffold, the preparation process needs to be strictly controlled. First, the magnesium oxide nanoparticles, gelatin and L-polylactic acid solution need to be stirred overnight (generally for more than 12 hours) to prevent agglomeration between the nanoparticles and the gelatin. As shown in Comparative Example 1, when the mixed solution was stirred for only 8 hours, some gelatin and nanoparticles clogged the nozzle during the spinning process due to agglomeration.

[0015] Secondly, strict control of electrospinning process parameters is required. For example, in Comparative Example 2, the spinning solution containing 1.05g of Gelatin / PLLA / MgO NPs was subjected to a voltage of 20kV. The Taylor cone range was large, resulting in scattered fibers without aggregation, making it difficult to collect the nanofiber membrane. Ultimately, only 0.6g of fiber membrane was collected.

[0016] Third, the pulverization time and intensity of the nanofiber membrane must be strictly controlled. Different intensities and times can lead to differences in fiber length, which can affect the scaffold structure. For example, in Comparative Example 3, after pulverization in a steel ball ultrasonic pulverizer for 5 minutes, large clumps of fiber membrane were visible on the macroscopic surface, clogging the pipette tip during aspiration. The resulting scaffold was clearly demarcated from the surrounding clumps, making the overall scaffold fragile. Microscopically, the fibers were not broken, but rather arranged in a disordered stack, lacking pores.

[0017] Fourth, the settling time must be strictly controlled. For example, in Comparative Example 4, where the short fiber suspension was dispensed without settling and then placed at -80°C overnight, the water contact angles on both sides of the scaffold were found to be approximately 110 degrees, indicating that the scaffold was hydrophobic. In Comparative Example 5, where the short fiber suspension was dispensed and allowed to settle for 30 minutes before being placed at -80°C overnight, the overall thickness of the scaffold decreased from 3 mm to 1-1.5 mm, and the water contact angles on both sides were approximately 125-130 degrees, indicating that the scaffold was hydrophobic. Microscopically, gaps between the fibers were barely visible.

[0018] Furthermore, the weight ratio of the magnesium oxide nanoparticles, gelatin and poly (L-lactic acid) in step (1) is 1:16:4.

[0019] Furthermore, the magnesium oxide nanoparticles in step (1) are prepared by first heating and stirring magnesium chloride, glacial acetic acid and ammonia water to obtain a colloidal liquid, and then drying and calcining the colloidal liquid.

[0020] Furthermore, in step (1), gelatin and poly (L-lactic acid) are dissolved in hexafluoroisopropanol.

[0021] Furthermore, the solvent used for the short fiber suspension in step (2) is tert-butanol, and the mass volume ratio of the short fiber suspension is 2%.

[0022] Furthermore, the freeze drying in step (2) is to store the short fiber suspension at -80°C overnight and then freeze dry it for 24 hours.

[0023] Furthermore, the thermal crosslinking in step (2) is performed at 180° C. for 2 hours.

[0024] A second object of the present invention is to provide a magnesium oxide-loaded asymmetric short fiber aerogel scaffold obtained by the preparation method described above.

[0025] A third object of the present invention is to provide an application of the above-mentioned magnesium oxide-loaded asymmetric short fiber aerogel scaffold in the preparation of wound repair medical materials.

[0026] On the other hand, the present invention also provides the use of the above-mentioned magnesium oxide-loaded asymmetric short fiber aerogel scaffold in the preparation of a medical material for promoting wound repair in diabetic patients.

[0027] The beneficial effects of the present invention are as follows:

[0028] The present invention develops an asymmetric short fiber gelatin / polylactic acid aerogel scaffold (GP@MgO) loaded with magnesium oxide nanoparticles. This short fiber aerogel scaffold solves the two-dimensional longitudinal collapse problem of electrospinning, realizes a spatial three-dimensional biomimetic structure, provides more adhesion sites for cells, and can slowly absorb tissue exudate. Under the action of gravity, the short fibers form an asymmetric density at both ends, which helps to manage exudate. At the same time, this aerogel can relieve over-activated inflammation, improve cell energy supply defects, accelerate neovascularization, and slow down the aging process of tissues near the wound. In addition, GP@MgO regulates macrophage polarization and reduces the release of aging-related secretory phenotypes, achieving accelerated angiogenesis and re-epithelialization in a full-thickness skin model of diabetic rats. Therefore, the short fiber GP@MgO aerogel of the present invention provides a means of regulating biochemical functions and physiological responses for rapidly closing diabetic wounds through metal immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Figure 3. Morphology and properties of GP@MgO aerogel; a) Digital illustration of the key steps in aerogel preparation; b) Scanning electron microscope images of NF cloth substrate, GP aerogel, and GP@MgO aerogel; c) Aerogel density analysis; d) Fiber diameter analysis; e) Aerogel porosity analysis; f) Element distribution diagram of GP@MgO aerogel by energy spectrum analysis; g) XRD pattern; h) FTIR spectrum; i) Release of magnesium ions from GP@MgO aerogel; j) Water contact angle analysis; k) Young's modulus; l) SEM images of different aerogel surfaces; m) Water contact angle and analysis of different aerogel surfaces; n) 3D image reconstructed by laser scanning microscopy; (ns indicates no significant difference, ****P<0.0001).

[0030] Figure 2GP@MgO aerogel has excellent biocompatibility, can promote cell function, and can delay the aging of endothelial cells; a) Live / dead cell staining at 1, 4, and 7 days (scale bar: 100 μm); b) EDU staining at 1, 4, and 7 days (scale bar: 100 μm) and k) statistical analysis; c) cell migration (scale bar: 200 μm) and l) statistical analysis; d) cytoskeleton staining (scale bar: 20 μm); e) Immunofluorescence staining of vascular endothelial growth factor (scale bar: 50 μm) and m) statistical analysis; f) Tube formation (scale bar: 200 μm) and n / p) statistical analysis of angiogenesis images; g) SA-β-galactose staining (scale bar: 200 μm); h) p21 immunofluorescence staining (scale bar: 50 μm) and q) statistical analysis; i) DCFH-DA staining (scale bar: 200 μm) and r) statistical analysis; j) JC-1 staining (scale bar: 50 μm); (ns indicates not significant difference; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0031] Figure 3 GP@MgO aerogel has excellent biocompatibility, can promote cell function, and can delay the aging of fibroblasts; a) Live / dead cell staining on day 1, day 4, and day 7 (scale bar: 100 μm); b) EDU staining on day 1, day 4, and day 7 (scale bar: 100 μm) and j) statistical analysis; c) cell migration (scale bar: 200 μm) and k) statistical analysis; d) cytoskeleton staining (scale bar: 50 μm); e) Immunofluorescence staining of collagen III (scale bar: 50 μm) and l) statistical analysis; f) SA-β-gal staining (scale bar: 100 μm); g) immunofluorescence staining of p21 (scale bar: 50 μm) and n) statistical analysis; h) DCFH-DA staining (scale bar: 200 μm) and m) statistical analysis; i) JC-1 staining (scale bar: 50 μm) and o) statistical analysis; (ns, not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0032] Figure 4GP@MgO aerogel can regulate the phenotype of macrophages and the expression of extracellular matrix proteins; a) Immunofluorescence staining of CD86 (scale bar: 100 μm) and g) statistical analysis; b) Immunofluorescence staining of CD206 (scale bar: 100 μm) and h) statistical analysis; c) flow cytometry histogram analysis (CD86) and i) quantitative analysis; d) flow cytometry histogram analysis (CD206) and j) quantitative analysis; e) Immunofluorescence staining of tumor necrosis factor-α (scale bar: 50 μm) and k) statistical analysis; f) Immunofluorescence staining of tumor necrosis factor-α (scale bar: 50 μm) and l) statistical analysis; (ns indicates no significant difference; *P<0.05, **P<0.01, ***P<0.001).

[0033] Figure 5 Figure 3. Magnesium oxide-based gel-like substances promote wound healing in diabetic rats; a) Representative images of wound closure in diabetic rats at different time points; b) Digital reconstruction of wound area; c) Percentage of wound area; d) Wound area on day 21; e) Staining results (scale bars: 200 μm and 50 μm); f) Mason trichrome staining (scale bars: 200 μm and 50 μm); (ns indicates no significant difference;) *P < 0.05, **P < 0.01, ***P < 0.001.

[0034] Figure 6 GP@MgO aerogel can reduce inflammation, delay aging and promote epithelial tissue regeneration; a) Immunofluorescence staining shows iNOS (green) and Arg 1 (red), and fg) quantitative analysis (scale bar: 40 μm); b) Immunofluorescence staining shows CK 14 (red) and j) quantitative analysis (scale bar: 200 μm and 50 μm); c) Immunohistochemical staining shows IL 1β and h) quantitative analysis (scale bar: 50 μm); d) Immunohistochemical staining shows IL 4 and i) quantitative analysis (scale bar: 50 μm); e) Immunofluorescence staining shows p21 (red) and k) quantitative analysis (scale bar: 200 μm); (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0035] Figure 7GP@MgO aerogel can promote collagen deposition and neovascularization; a) Immunofluorescence staining of collagen I (red) and e) quantitative analysis (scale bar: 200 μm); b) Immunofluorescence staining of type III collagen (red) and f) quantitative analysis (scale bar: 200 μm); c) Immunofluorescence staining of CD31 (red) and g) quantitative analysis (scale bar: 200 μm); d) Immunofluorescence staining of α-SMA (red) and h) quantitative analysis (scale bar: 200 μm); (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0036] Figure 8 Schematic diagram of the preparation process of GP@MgO short fiber aerogel and its therapeutic measures for immune-induced aging in diabetic rats and its effect on promoting wound healing.

[0037] Figure 9 Figure 3. Magnesium oxide nanoparticle morphology: a) Representative scanning electron microscopy image; b) Nanoparticle size distribution; c) EDS magnesium and oxygen elemental maps.

[0038] Figure 10 To characterize: a) elemental composition and image combination of micro / nanofiber mats; b) laser scanning microscope electron image and 3D reconstruction image.

[0039] Figure 11 Identification and demonstration of the type 1 diabetic rat model: a) blood glucose change curve of rats (measured by blood glucose meter); b) body weight change curve of rats; c) representative HE and fluorescence staining of the pancreas of type 1 diabetic rats; d) enzyme-linked immunosorbent assay for glucose; e) enzyme-linked immunosorbent assay for insulin; (ns, not significant). Figure 12 Biosafety testing of aerogels, such as HE staining of key organs, including heart, liver, spleen, lung, and kidney (scale bar: 200 μm).

[0040] Figure 13 GP@MgO aerogels enhance the nascent and ordered deposition of collagen in vivo: a) Immunofluorescence staining of Col I (red, scale bars: 200 μm and 50 μm); b) Immunofluorescence staining of Col III (red, scale bars: 200 μm and 50 μm).

[0041] Figure 14 GP@MgO aerogels accelerated the formation of new blood vessels in vivo: a) Immunofluorescence staining of CD31 (red, scale bars: 200 μm and 50 μm); b) Immunofluorescence staining of α-SMA (red, scale bars: 200 μm and 50 μm). DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the following embodiments. It is necessary to point out that the following embodiments are only used to explain and illustrate the present invention and are not intended to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still fall within the scope of protection of the present invention.

[0043] Example 1

[0044] 1. Experimental Materials and Methods

[0045] 1. Experimental Materials

[0046] Poly(L-lactic acid) [PLLA] was purchased from Jinan Dagang Co., Ltd. (Jinan, China); gelatin was purchased from Thermo Fisher Scientific (Shanghai, China); 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and tert-butyl alcohol were purchased from Shanghai Aladdin Biochemical Reagent Co., Ltd. (Shanghai, China); and cell culture reagents were provided by Qiagen (Carlsbad, California).

[0047] 2. Preparation of GP@MgO aerogel

[0048] (1) Preparation of magnesium oxide nanoparticles: Prepare 100 ml of 0.1 M magnesium chloride solution, add 200 μl of glacial acetic acid, heat the solution to 50°C, add ammonia water dropwise while stirring to adjust the pH to 9, and gel the solution. Then, dry the solution in a 100°C forced air drying oven. Then, dissolve the resulting powder in an appropriate amount of anhydrous ethanol and dry it in a 60°C forced air drying oven. The resulting powder is calcined in a muffle furnace with a heating rate of 1°C, a calcination temperature of 500°C in air, and a calcination time of 1.5 hours. The resulting white powder is magnesium oxide nanoparticles (MgO NPs).

[0049] (2) Preparation of aerogel: Aerogel is prepared by steps including electrospinning, pulverization, freeze drying and cross-linking. The specific method is as follows:

[0050] First, a Gel / PLLA-MgO (GP@MgO) micro / nanofiber mat (NF mat) was prepared as follows: 800 mg of gelatin, 200 mg of poly (L-lactic acid) (PLLA), and 50 mg of MgO nanoparticles were dissolved in 10 mL of hexafluoroisopropanol (HFIP) to prepare an electrospinning solution. The prepared solution was stirred overnight at 20°C and then loaded into a 10 mL syringe for electrospinning. The solution was continuously pumped at a flow rate of 3 mL / h using a syringe pump (Foshan Nanofiber Laboratory, Foshan, China) and electrospun between a spinneret (22-gauge needle) using a high-voltage generator (Tianjin Dongwen High Voltage, China) at a potential of 17 kV (17-19 kV was acceptable). The nanofiber membrane was collected using a rotating base at 300 rpm. The distance between the needle and the collector was 15 cm. The GP@MgO fiber membrane was vacuum-dried to remove trace amounts of solvent.

[0051] Subsequently, a suspension of GP@MgO short fibers was prepared using a grinder (Shanghai Jingxin, Shanghai, China) in a tert-butanol solution. The sheared fiber mat was ground in tert-butanol at a mass volume fraction of 2% (w / v) at 25 Hz for 10 minutes. The ground suspension was transferred to a 24-well plate, allowed to stand at 25°C for 15 minutes, and then stored in a -80°C freezer overnight. After thorough drying for 24 hours (using a Folgers dryer, Beijing, China), the sample was oven-stabilized at 180°C for 2 hours to facilitate cross-linking. GP aerogels were synthesized using the above steps. The cross-linked aerogels were treated under vacuum for 24 hours and then subjected to double-sided UV irradiation for 6 hours before use in in vitro cell culture and in vivo animal studies.

[0052] 3. Characterization of materials

[0053] The aerogel morphology was observed using a scanning electron microscope (SEM, TESCAN MIRA LMS, Czech Republic). The average fiber diameter and porosity were measured using ImageJ software. The mass of the aerogel was measured using an electronic balance (accuracy 0.0001 g), and its diameter and height were measured using a vernier caliper (accuracy 0.02 mm).

[0054] Calculate the volume and density of the aerogel using the following formula:

[0055] V=πr 2 h, ρ(g·cm -3 )=m / V

[0056] where ρ is the density, V, r, h, and m are the volume, radius, height, and mass of the aerogel, respectively.

[0057] GP, GP@MgO, and MgO nanoparticles were characterized using Fourier transform infrared spectroscopy (FTIR, Thermo Fisher Scientific iN10, USA) and X-ray diffraction (XRD, Bruker D8 Advance, Germany). The water contact angles of GP and GP@MgO aerogels were measured using a water contact angle analyzer (WCA, Dingsheng JY-82C, China). Compression tests were performed on GP and GP@MgO aerogels using a universal testing machine (MTS CMT6103, USA). The aerogels were cylindrical, 3 mm high and 10 mm in diameter. Stress changes under varying strain levels were recorded, and Young's modulus was calculated. The magnesium ions released from the GP@MgO aerogels were quantitatively analyzed using inductively coupled plasma optical emission spectroscopy (ICP-OES; Agilent 5110, USA). The aerogels were placed in 5 mL of double-distilled water, and 1 mL of the liquid was sampled at each time point for testing at 37°C.

[0058] A laser scanning microscope (Keyence VK-X1000, Japan) was used to detect the different morphologies and roughness on both sides of the aerogel, and three-dimensional reconstruction was performed using a multi-file analyzer.

[0059] 4. Cell culture

[0060] Human umbilical vein endothelial cells (HUVECs) and 208F cells were cultured in Dulbecco's modified Eagle's medium (DMEM). Bone marrow-derived mononuclear phagocytes (BMDMs) were isolated according to the literature and cultured in minimum essential medium (MEM) supplemented with recombinant rat macrophage colony-stimulating factor (M-CSF; PeproTech, USA). The culture medium was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and replaced every 2 days. In addition, 100 ng / mL lipopolysaccharide (LPS; Sigma) was added to the culture medium in all in vitro experiments to simulate an inflammatory environment.

[0061] 5. Cell biocompatibility and proliferation analysis

[0062] HUVECs or 208F cells were plated at 2.5 × 10 4Cells were seeded at a density of 100 cells per well in 12-well plates and co-cultured with one aerogel per well for 1, 4, and 7 days. The cells were stained with live and dead cell stains. The cells were incubated in a working solution of 2 μM anthocyanin AM and 4.5 μM PI (Yesain Biotechnology Co., Ltd., China) for 15 minutes and observed under a fluorescence microscope. In addition, the culture medium was replaced with a working solution of 10 μM 5-ethynyl-2-deoxyuridine (EDU, APE BIO, K1175, K1075, USA) and incubated with human umbilical vein endothelial cells (HUVECs) for 2 hours and 208Fs for 4 hours at 37°C. After fixation and permeabilization, the click reaction was performed as described and stained with 5 μg / mL Hoechst 33342. Wash three times with PBS containing 3% BSA between each step.

[0063] HUVECs and 208Fs were cultured at 5×10 4 Cells were seeded at a density of 100 cells / mL in 12-well plates and cultured for 3 days. After fixation with 4% formalin for 15 minutes, the cells were blocked with 10% goat serum at 20°C for 2 hours, and then incubated with VEGF (1:200, affinity type) and Col III (1:200, affinity type) at 4°C overnight. The cells were then incubated with fluorescein isothiocyanate-labeled goat anti-rabbit IgG (H+L) (1:1000, APE BIO) and Cy3-labeled goat anti-rabbit IgG (H+L) (1:1000, APE BIO) for 2 hours. The cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, 10 μg / mL, Biosharp) for 10 minutes. Images were acquired using a fluorescence microscope under various excitation conditions.

[0064] 6. Scratch and tube formation experiments

[0065] HUVECs and 208Fs cells were cultured at a rate of 1.5 × 10 5 Cells were seeded at a density of 100 μL in a 6-well plate and incubated at 37°C for 24 hours. A 200 μL tip was used to scratch the bottom of the plate in parallel. The culture medium was replaced with 2% FBS to minimize the effects of cell proliferation. The migration area of ​​the scratch was observed at 0, 12, and 24 hours, and photographed using a fluorescence microscope.

[0066] Apply 100 μL of Matrigel wash solution evenly on a pre-cooled 24-well plate and solidify at 37°C for 30 minutes. Seed 5×10 4 HUVECs were plated and incubated for 4 hours. The cells were stained with Calcein AM for 10 minutes, and tube formation was observed using a fluorescence microscope.

[0067] 7. Cellular aging and mitochondrial function

[0068] HUVECs and 208Fs cells were plated at 5 × 10 4 The cells were seeded in 12-well plates at a density of 100 cells / well and cultured for 3 days. The cells were stained for senescence-associated β-galactosidase (SA-β-gal) using a staining kit produced by Biothem.

[0069] First, cells were fixed with β-galactosidase fixative for 15 minutes and then incubated with β-galactosidase working solution overnight at 37°C. Next, samples were washed with 70% ethanol to remove crystals and subsequently photographed. Furthermore, before photographing, slides were fixed, mounted, and stained with p21 (1:200; affinity). Observation was then performed under a fluorescence microscope.

[0070] HUVECs and 208Fs cells were seeded in 12-well plates at a density of 5 × 10 cells per well. 4 Cells were cultured for 3 days. The cells were incubated with mitochondrial membrane potential working solution (from Sun Biotech, China) or 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) working solution (also from Sun Biotech, China) for 20 minutes and then imaged using a fluorescence microscope.

[0071] 8. Macrophage phenotype

[0072] Cells isolated from rat bone marrow were cultured in complete medium with 20 ng / mL M-CSF for 24 hours. The supernatant was transferred to a new cell culture flask and cultured for another 3 days. Macrophages were seeded in 12-well plates and co-cultured with GP and GP@MgO aerogels for 24 hours at 37°C. After fixation and restraint, cells were incubated overnight at 4°C with CD86 (1:200, Proteintech), CD206 (1:200, Proteintech), TNF-α (1:200, Avidin), or IL-10 (1:200, Avidin). F-actin was stained with rhodamine-labeled phalloidin (1:200, Sun Biotech) and FTIC-labeled phalloidin (1:200, Sun Biotech) for 30 minutes, respectively. Cell nuclei were stained with a nuclear dye and stained with DAPI for 10 minutes. Images were acquired using a fluorescence microscope at different fluorescence excitation wavelengths. Similarly, cells co-cultured with aerogels for 24 hours were harvested and macrophage polarization was assessed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). The samples were incubated on ice in the dark for 30 minutes using antibodies against CD86 (1 μg each, Invitrogen) and CD206 (1 μg each, Bioss).

[0073] 9. Animal research

[0074] All protocols used for in vivo experiments were performed in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. The Animal Research Committee of Bengbu Medical University approved all procedures related to animal experiments (approval number: 2024602). Male Sprague-Dawley (SD) rats (weighing 200–240 g) were purchased from the Zhejiang Laboratory Animal Center (Hangzhou, China). After a 12-hour fast, these rats were intraperitoneally injected with 1% streptozotocin (STZ, 55 mg / kg, Sorbo Biological Products). One week after feeding, STZ-injected rats had random blood glucose levels of 16.7 mmol / L, accompanied by weight loss, polydipsia, and polyuria. These rats were considered successful type 1 diabetes models and used in subsequent experiments. The diabetic rats were randomly divided into three groups: control, GP, and GP@MgO. After anesthesia, three 1 × 1 cm circular full-thickness skin defects were created along the dorsal midline of the rat spine. Wound healing was recorded on days 0, 3, 7, 14, and 21, and samples were collected at these time points.

[0075] Wound size was calculated using the following formula:

[0076] Wound size (%) = S t / S0×100%

[0077] Among them, S0 is the wound area on day 0, S t The wound areas were on days 3, 7, 14, and 21. On day 21, serum samples and organ samples including heart, liver, spleen, lung, kidney, and pancreas were collected from the rats to verify biosafety and the presence of type 1 diabetes.

[0078] 10. Histology

[0079] The isolated skin and organ tissues were immersed in 4% paraformaldehyde (PFA) for 24 hours, then dehydrated through an ethanol gradient, embedded in paraffin, and cut into 7-μm sections. Hematoxylin-eosin (HE) staining and Masson's trichrome staining were used to assess diabetic wound healing. p21 (1:200, affinity) was used to assess the senescence process. Platelet endothelial cell adhesion molecule-1 (CD31, 1:200, Proteintech) and α-smooth muscle actin (α-SMA, 1:100, affinity) were used as vascular markers to count the number of new vessels and assess the extent of vascular remodeling. Collagen deposition was assessed using collagen I (COL I, 1:100, affinity) and collagen III (COL III, 1:100, affinity). Cytokeratin 14 (CK14, 1:200, Proteintech) was used as an epithelial cell marker to assess the degree of re-epithelialization. Inducible nitric oxide synthase (iNOS, 1:200, Proteintech) and arginase-1 (Arg-1, 1:400, Proteintech) were used as macrophage markers to assess macrophage polarization. IL-1β and IL-4 were used as inflammatory cytokines to assess inflammation. Insulin (1:400, Servicebio) and glucagon (1:400, Servicebio) were used as markers of pancreatic A cells and B cells, respectively, to assess the establishment of the diabetic model.

[0080] 11. Statistical analysis

[0081] All data are presented as mean ± SD. Statistical analysis was performed using Student's t-test and analysis of variance using GraphPad Prism (version 8.0.1). Measurements were acquired from images using ImageJ. Graphs were created using GraphPad Prism and Origin Pro (version 8.5). The significance levels were set as follows: ns (no significant difference, p > 0.05), * (p < 0.05), ** (p < 0.01), *** (p < 0.001).

[0082] 2. Experimental Results and Discussion

[0083] 1. Morphology and characterization of GP@MgO aerogel

[0084] Relevant properties of magnesium oxide nanoparticles such as Figure 9 Scanning electron microscopy (SEM) images showed that the particle size was approximately 40 nm, and energy dispersive X-ray spectroscopy (EDS) revealed the presence of an overlap of magnesium and oxygen elements.

[0085] GP@MgO short-fiber aerogels with extracellular matrix structure were prepared by electrospinning, crushing, freeze-drying and crosslinking. Two-dimensional micro / nanofiber webs were prepared by electrospinning Gel / PLLA / MgO nanoparticle solution in HFIP. Then, the fiber webs were crushed in t-butyl alcohol solution. Finally, they were dried and crosslinked to stabilize the short-fiber aerogel structure Figure 1 The NF web was tightly arranged by overlapping layers, which could be seen from the EDS images showing typical fiber layers and morphology Figure 2 Figure 10 The SEM data showed that the upper surface of GP and GP@MgO short-fiber aerogels destroyed the regular network structure of the fiber web, presenting irregular and folded micro / nanofibers with loose and porous structure Figure 2 The fiber diameter of the short-fiber aerogels was about 1 micron, with extremely low density and high porosity; there was no significant difference between GP and GP@MgO Figure 1 Figure 1 EDS analysis showed the distribution of MgO nanoparticles in the GP@MgO aerogel

[0086] In addition, XRD analysis further confirmed the presence of MgO nanoparticles in the fibers Figure 1 Figure 1 The FTIR spectra of MgO nanoparticles, GP aerogel and GP@MgO aerogel are shown in Figure 1 Due to the sensitivity of MgO nanoparticles to water absorption and hydrolysis, which would lead to the formation of magnesium hydroxide, the absorption peak of hydroxyl at 3700 nm can be used as a marker. However, due to the relatively low concentration of MgO nanoparticles, this peak was not observed in the aerogel. The mechanical properties of the short-fiber aerogels and the release of Mg ions are shown in Figure 1

[0087] Figure 1 Figure l shows scanning electron microscope (SEM) images of different sides of the aerogel, which show different fiber densities and porosities. The top fibers are sparse and porous, while the bottom fibers are dense and stacked. Figure 1 ​​​​The figure shows the hydrophilicity and hydrophobicity of the short fiber aerogel based on the water contact angle. It is worth noting that there is a difference in the degree of hydrophilicity between the top and bottom of the aerogel: the bottom fibers are flat, but the water contact angle exceeds 120°, while the top surface of the aerogel is uneven, with a water contact angle of less than 90°. The 3D reconstructed laser scanning microscopy images show the morphology and roughness of different surfaces of the aerogel ( Figure 1 The top image shows numerous pores and extensive longitudinal extensions, while the bottom image illustrates the relatively flat shape of the short fibers after they have been stacked down under gravity.

[0088] Biomaterial scaffolds should align with the natural extracellular matrix and adapt to the complexity of local defects to promote tissue regeneration. Conventional electrospun membranes are composed of tightly packed fibers with small pore size and low porosity, which are not conducive to cell growth and therefore unsuitable for biomedical applications. Short-fiber aerogels exhibit a three-dimensional porous structure by reshaping the structure of pulverized fibers. The short fibers initially undergo physical entanglement, enhancing mechanical bonding. Simultaneously, the abundant amino groups in gelatin and the carboxyl groups in PLLA promote the formation of peptide bonds through thermal crosslinking, further enhancing mechanical properties and stability. Furthermore, the different hydrophilicity observed on both sides of the sample can be attributed to the uneven fiber density resulting from gravitational settling of the short fibers within the trimer (after pulverization and distribution, a butanol solution is used. The resulting fiber density variation affects surface tension and pore size, which in turn influences capillary forces. Liquid is drawn in through capillary forces and gradually absorbed by the aerogel, maintaining a dry wound bed environment. The exposed portions evaporate absorbed liquid and increase the liquid absorption rate). Therefore, the GP@MgO short fiber aerogel is able to absorb and evaporate the exudate tissue fluid in the wound bed over time while gradually exposing the loaded MgO nanoparticles.

[0089] 2. Biocompatibility of GP@MgO aerogel in vitro

[0090] Aerogels, composed of intertwined nanofibers, have a unique structure that enables them to reproduce the configuration of the natural extracellular matrix (ECM), thereby regulating various cellular activities, including cell proliferation, migration, and secretion. Figure 2 A and Figure 3 As shown in (a), the cell number increased over time, and the cell morphology was stretched, especially in the GP@MgO group. Live / dead cell staining showed that there were almost no dead cells. The number of proliferating cells on days 1, 4, and 7 was assessed by EDU staining. The fluorescence level of GP@MgO was higher than that of the control group and GP group, indicating that cell proliferation was promoted ( Figure 2 In b and k, Figure 3 The effect of GP@MgO aerogel scaffold on cell migration was evaluated by scratch test ( Figure 2In the middle, c and i, Figure 3 c and k). At 12 and 24 hours, GP@MgO showed higher cell migration rate to the damaged area than other groups. Nuclear skeleton staining showed that the cytoskeleton was fully unfolded, indicating that the aerogel scaffold had no cytotoxicity ( Figure 2 Zhongd and Figure 3 In addition, GP@MgO significantly enhanced the expression of VEGF ( Figure 2 e and m). Comparison of the nodes and tube lengths of different groups After 4 hours of treatment with different types of aerogels, it was found that the formation of GP@MgO was significant, while the control group showed almost no formation of tubular structures ( Figure 2 f, n, o, and p in the figure). In addition, GP@MgO enhanced the expression of Col III. In conclusion, GP@MgO aerogels improved the physiological activities of HUVEC and 208F cells and provided the possibility for angiogenesis and collagen deposition in vivo.

[0091] 3. Delay of cell senescence and regulation of macrophage polarization in GP@MgO aerogels under in vitro conditions

[0092] Fluctuations in blood glucose levels in diabetic patients lead to an imbalanced microenvironment, which can trigger oxidative stress, collagen synthesis, angiogenesis, cell proliferation, and other adverse conditions in tissue regeneration. Sustained increases in blood glucose in the body cause acidic changes in the surrounding microenvironment, prompting the release of factors related to the inflammatory cytokine family and deformation of the extracellular matrix. This persistent inflammatory state impairs mitochondrial function and delays the clearance of reactive oxygen species (ROS). Furthermore, the inflammatory response at the site of the wound defect further exacerbates the already hostile microenvironment, thereby promoting immune-induced aging. Thus, the hyperglycemic environment associated with diabetes prevents the body from effectively responding to the consequences of external trauma.

[0093] We investigated the senescent phenotype of human umbilical vein endothelial cells (HUVECs) and 208Fs cells to address this question. Under LPS-induced inflammation, the cells showed increased SA-β-Gal activity ( Figure 2 Medium g and Figure 3 However, the addition of GP membrane gel did not alleviate this phenomenon. On the contrary, GP@MgO led to a significant downregulation of expression. Similarly, GP@MgO membrane gel ( Figure 2 h and q, Figure 3 Figures g and n) show that the expression of the aging biomarker p21 is significantly reduced. Studies on mitochondrial function further reveal the cause of cell aging. Based on DCFH-DA ( Figure 2 In i and r, Figure 3 h and m), ROS fluorescence expression was inhibited and significantly lower than that of other groups. The examination of mitochondrial membrane potential yielded consistent results ( Figure 2 Zhongj and Figure 3 (i) JC-1 staining revealed a significant depolarization of the mitochondrial membrane potential following immune induction, accompanied by a significant increase in JC-1 monomer levels. However, repolarization was observed after GP@MgO treatment, indicating that inflammation-induced mitochondrial function was restored.

[0094] In the body's immune response, we further verified that GP@MgO aerogel regulates immune function through its effects on macrophage polarization and inflammatory cytokine secretion. Immunofluorescence staining was performed by labeling M1 and M2 cell surface markers (CD86 and CD206) after inducing bone marrow-derived macrophage (BMDM) differentiation. Figure 4 (a, b, g, and h). The LPS-stimulated control group showed an increase in the proportion of M1 macrophages, while M1 macrophages co-cultured with GP aerogels still highly expressed. However, GP@MgO treatment reduced the number of M1 macrophages and increased the expression of M2 markers. In addition, flow cytometry comparison showed that fluorescence expression showed a consistent trend, and the expression of CD206 in the GP@MgO group was significantly stronger than that in the other groups ( Figure 4 c, d, i and j). In addition, if Figure 4 As shown in Figures e and f, consistent results were obtained by comparing IL-10, a marker representing M2, and TNF-α, a marker representing the extracellular matrix (SASP). GP@MgO treatment significantly increased the expression level of anti-inflammatory factors. It significantly hindered the inflammatory response. Compared with the control group and the GP group, the secretion of SASP (see Figure 4 From the above results, it can be inferred that GP@MgO significantly enhanced the trend of macrophage polarization toward M2 type in inflammatory environment and improved the microenvironment that is not conducive to immune-induced aging.

[0095] The magnesium oxide nanoparticles released from the aerogel react with water to form magnesium hydroxide (Mg(OH)2), which has a certain solubility in water and will completely decompose into magnesium ions Mg 2+ , water molecules and hydroxide ions (OH - ). The high glucose microenvironment in diabetes leads to chronic inflammation, which triggers inflammation and accelerates the aging process of the organism. Dissociated OH - Can effectively neutralize hydrogen ions (H + ) and lactate produced by glycolysis regulate the pH of the local microenvironment under conditions of traumatic hypoxia. Studies have shown that elevated pH within the microenvironment can effectively downregulate aging markers (p16, p21) through the PI3K / AKT / mTOR pathway. The sustained decrease in hydroxide ion levels leads to the sustained hydrolysis of magnesium oxide, thereby maintaining the release of magnesium ions.

[0096] 4. Assessment of diabetic wound healing

[0097] Based on the results of in vitro experiments showing that GP@MgO aerogel can regulate cell proliferation, promote angiogenesis and macrophage polarization, a full-thickness diabetic wound model was established to further verify the effect of aerogel on diabetic wound healing in vivo. In the type 1 diabetes model, the random blood glucose level of diabetic rats during the healing period was always higher than 16.7mmol / L, and the body weight decreased significantly in the early stage ( Figure 11 In a and b), pancreatic islets are found in Figure 11 Middle c.

[0098] A digital camera (produced by Canon, Japan) was used to record the wound healing process. Figure 5 As shown in Figure a. During the treatment period, the degree of wound healing varied between different groups exposed to the same feeding environment. The wound area of ​​the wounds treated with GP@MgO aerogels was smaller than that of the wounds treated with the control group and GP aerogels at each time point, and the healing process was significantly faster. On the 3rd day, the untreated wounds appeared moist, and obvious yellow exudate was visible; on the 7th day, the wounds had not yet completely scabbed and exudate was still present. The wound bed treated with GP and GP@MgO was observed to be dry on the 3rd day, and the yellow exudate was adsorbed on the surface of the GP aerogel. On the 7th day, the scab completely covered the wound. The wounds treated with GP@MgO on the 21st day showed signs of high healing, and the degree of healing increased as the treatment progressed. In contrast, the control group still had a larger wound area. In order to visualize the changes in the wounds, the dynamic healing of each group throughout the healing process was digitally reconstructed ( Figure 5 b). Quantitative results at each time point ( Figure 5 Figures c and d) show the changes during the healing process. The residual wound area in the GP@MgO group was smaller than that in the GP and control groups, indicating that MgO nanoparticles have a positive effect on diabetic wound healing.

[0099] HE staining was performed to further evaluate the in vivo healing ( Figure 5 (e) In each group, wound length gradually decreased with prolonged healing time. Granulation tissue thickness gradually increased over time. While the control and GP groups showed minimal tumor tissue, the GP@MgO group displayed abundant granulation tissue at day 7. However, the thickness of the GP@MgO group decreased compared to the previous measurement at day 21. These results indicate that wounds in the GP@MgO membrane gel group achieved epithelial regeneration and initiated granulation tissue remodeling, while wound healing in the control and GP groups was delayed. Furthermore, at day 21, a more complex epidermal structure, including skin appendages, was observed in the GP@MgO group compared to the other groups.

[0100] In addition, the hair follicle regeneration of each group on the 21st day was analyzed by Masson staining ( Figure 5 f) was observed (the control group did not form complete hair follicles on the 14th day). In contrast, the GP@MgO group showed mature and numerous hair follicles. The difference in the number of hair follicles between the groups was more obvious on the 21st day. These results indicate that GP@MgO aerogel can significantly accelerate the wound healing process in diabetic patients and shorten the healing time. The in vivo biosafety of GP and GP@MgO was demonstrated by biopsy of the heart, liver, spleen, lung and kidney of rats ( Figure 12 ), without causing obvious damage to major organs.

[0101] 5. Cell aging and re-epithelialization process in vivo

[0102] The best way to manage diabetic wounds is to control the progression of the inflammatory phase. Macrophages play a key role in the inflammatory phase and are important indicators for initiating the regenerative process and are a key factor in predicting wound healing outcomes. Macrophages were detected by immunofluorescence staining for iNOS and Arg-1 in all groups on days 3 and 7. Figure 6 (a). At the initial stage of the inflammatory process (day 3), the iNOS level in the control group was significantly higher than that in the GP@MgO group, indicating enhanced inflammatory activity. However, the GP@MgO group had a significant number of M2 macrophages at both time points, indicating that MgO nanoparticles promoted the transition of macrophages from M1 to M2. Notably, GP membrane aerogel did not significantly reduce the number of M1 macrophages. Instead, it increased the expression of Arg-1, ultimately causing the wound to move from the inflammatory stage to the proliferative stage ( Figure 6 Based on the above situation, we further evaluated the intensity of inflammatory response by immunohistochemical analysis of IL-1β and IL-4 ( Figure 6 (b, h and c, i). On day 7, expression of IL-1 (a pro-inflammatory marker) was highest in the control group and lowest in the GP@MgO group. Similarly, the GP@MgO group showed a similar pattern. Expression of IL-4 (an anti-inflammatory marker) was significantly higher than in both the control group and the GP film-like gel group. These results suggest that the GP@MgO film-like gel can effectively regulate the inflammatory phase of diabetic wounds.

[0103] To further verify the effect of controlling inflammation on wound tissue senescence, p21 staining was performed to assess the degree of senescence ( Figure 6(e and k) Compared with the control group, the p21-positive area was significantly reduced after GP@MgO treatment, indicating successful suppression of p21 expression, a senescence marker. Taken together, these in vitro and in vivo experiments demonstrate that the GP@MgO aerogel short fiber scaffold can improve the immune microenvironment in diabetic patients and rescue senescence through an inflammation-mediated pathway.

[0104] Cytokeratin (CK) is an important biomarker associated with epidermal differentiation and re-epithelialization. CK14 is an intermediate type I fibrokeratin family member that is expressed during the development of undifferentiated composite epithelium and forms basal keratin in composite squamous epithelial cytokeratin when paired with type II keratin CK5. Immunofluorescence staining of CK14 showed that in the new epidermis of the GP@MgO group, the expression of red fluorescent CK14 was strong at every time point ( Figure 6 (middle b). Meanwhile, on day 21, the epithelial thickness in the GP@MgO group was lower than that on day 14. This was accompanied by the appearance of several hair follicles, indicating that the wound had entered the remodeling phase. In contrast, the epithelial thickness in the control group continued to increase. These indicate that the re-epithelialization of the wound tissue was accelerated, and the healing characteristics of the GP@MgO group were enhanced compared with the control group and the GP group ( Figure 6 These findings were consistent with the results of HE and Masson staining.

[0105] 5. Collagen deposition and neovascularization in the body

[0106] Ordered collagen deposition contributes to the remodeling of the extracellular matrix (ECM) and wound healing. Immunofluorescence detection at different healing time points after different scaffold treatments was performed to determine the deposition and remodeling of Col I and Col III ( Figure 7 a and b, Figure 13 (a and b). On day 3, sporadic collagen formation was observed only in the GP@MgO group. Thereafter, the expression levels of all groups increased significantly. However, the GP@MgO group performed significantly better than the other groups. Mature bundles of collagen fibers were observed to show a balanced arrangement. This was especially true on days 14 and 21. Overall, the expression level of Col III was higher than that of Col I ( Figure 7 e and f).

[0107] The typical lack of angiogenesis in diabetic wounds is a major obstacle to normal healing. Immunofluorescence staining was used to observe angiogenesis, in which CD31 and α-SMA were used to mark new blood vessels and mature blood vessels, respectively. Figure 7 Middle C and Figure 14As shown in FIG. 3a, the control group did not show any new blood vessel formation at day 3, while the GP@MgO group showed angiogenesis. At day 7, tumor microvessels were observed in all groups. In the GP@MgO group, significantly better results were achieved in terms of the number of blood vessels and the size of the ducts. At day 14, both the GP and GP@MgO groups showed the appearance of small blood vessels and medium-sized blood vessels. At day 21, the newly formed capillaries in the GP@MgO group degenerated, leaving only medium-sized blood vessels. In contrast, the control group showed the presence of capillaries and small blood vessels, indicating that the wound had not yet entered the remodeling phase. Although the level of a-SMA was lower than that of CD31 at the same time, the trend was consistent with that of CD31 Figure 7 FIGS. 3d and h, Figure 14 FIG. 3b. In particular, at day 21, the diameters of the blood vessels labeled by CD31 and a-SMA were highly similar in the GP@MgO group, and the absence of capillaries further confirmed that the wound had entered the remodeling phase. These findings indicate that the GP@MgO-treated wounds followed the appropriate wound repair pathway over time.

[0108] III. CONCLUSION

[0109] The present application prepared a biomimetic three-dimensional magnesium oxide nanoparticle-loaded aerogel (GP@MgO aerogel) through the steps of electrospinning, crushing, freeze-drying, and cross-linking, to accelerate the healing of diabetic wounds and inhibit immune-induced senescence Figure 8 ). The GP@MgO aerogel effectively absorbed tissue exudates through its three-dimensional porous structure, thereby keeping the wound dry. This alleviated the prognosis of inflammatory senescence by modulating the macrophage phenotype and improving the inflammatory microenvironment, and improved the inflammatory microenvironment by releasing the internal magnesium oxide nanoparticles. In addition, the aerogel promoted angiogenesis, collagen deposition, and re-epithelialization, thereby significantly accelerating the healing of diabetic wounds. Therefore, the GP@MgO aerogel scaffold provided by the present application can provide a potential method for alleviating the inflammatory aging of chronic wounds and provide a new therapeutic means for the healing of diabetic wounds.

[0110] Comparative Example 1

[0111] The short-fiber aerogel scaffold was prepared according to the method of Example 1, except that the magnesium oxide nanoparticles, gelatin, and left-handed polylactic acid were stirred for only 8 hours to form the spinning solution, and there was a phenomenon that part of the gelatin and nanoparticles were blocked in the nozzle during the spinning process due to clumping.

[0112] Comparative Example 2

[0113] The short fiber aerogel scaffold was prepared according to the method of Example 1, except that the spinning solution containing 1.05 g of Gelatin / PLLA / MgO NPs was electrospun at a Taylor cone range of 20 kV voltage, and the phenomenon of fiber dispersion and non-aggregation occurred, making it difficult to collect the fiber membrane. Finally, only 0.6 g of fiber membrane was collected.

[0114] Comparative Example 3

[0115] The short fiber aerogel scaffold was prepared according to the method of Example 1, except that during the grinding of the short fiber suspension, a steel ball ultrasonic crusher was used to crush for 5 minutes at 30 Hz. Macroscopically, large clumps of fiber membrane were visible, which clogged the gun head during suction. The final scaffold formation was in clumps, with a clear boundary between the clumps and the surrounding, and the overall scaffold was fragile. Microscopically, the fibers were not broken, and the layers were arranged in disorder, lacking pore size.

[0116] Comparative Example 4

[0117] The short fiber aerogel scaffold was prepared according to the method of Example 1, except that instead of standing for 15 minutes, the short fiber suspension was directly divided and placed in -80 overnight. It was found that the water contact angle of both sides of the scaffold was about 110 degrees, and the overall scaffold was hydrophobic.

[0118] Comparative Example 5

[0119] The short fiber aerogel scaffold was prepared according to the method of Example 1, except that after the short fiber suspension was divided, it was allowed to settle for 30 minutes before being placed in -80 overnight. It was found that the overall thickness of the scaffold decreased from 3 mm to 1-1.5 mm, and the water contact angle of both sides was about 125-130 degrees, which was hydrophobic. Under a microscope, almost no gap was visible between the fibers.

Claims

1. A method for preparing an asymmetric short fiber aerogel scaffold loaded with magnesium oxide, characterized in that: The following steps are involved: (1) Mixing magnesium oxide nanoparticles with a solution of gelatin and L-polylactic acid and stirring for more than 12 hours to prepare an electrospinning solution; then electrospinning the electrospinning solution under the following conditions: voltage 17-19 kV, rotation speed 300 rpm, flow rate 3 mL / h, and collection distance 15 cm to obtain a nanofiber membrane; (2) The nanofiber membrane obtained in step (1) is cut into pieces and then prepared into a short fiber suspension. The short fiber suspension is ground at 25 Hz for 10 minutes, then allowed to settle for 15 minutes, freeze-dried, and thermally cross-linked to prepare a short fiber aerogel scaffold.

2. The preparation method according to claim 1, characterized in that The weight ratio of the magnesium oxide nanoparticles, gelatin and poly (L-lactic acid) in step (1) is 1:16:

4.

3. The preparation method according to claim 1, characterized in that The magnesium oxide nanoparticles in step (1) are prepared by first heating and stirring magnesium chloride, glacial acetic acid and ammonia water to obtain a colloidal liquid, and then drying and calcining the colloidal liquid.

4. The preparation method according to claim 1, characterized in that In step (1), gelatin and poly (L-lactic acid) are dissolved in hexafluoroisopropanol.

5. The preparation method according to claim 1, characterized in that The solvent used for the short fiber suspension in step (2) is tert-butanol, and the mass volume ratio of the short fiber suspension is 2%.

6. The preparation method according to claim 1, characterized in that The freeze drying in step (2) is to store the short fiber suspension at -80°C overnight and then freeze dry it for 24 hours.

7. The preparation method according to claim 1, characterized in that The thermal crosslinking in step (2) is carried out at 180° C. for 2 hours.

8. A magnesium oxide-loaded asymmetric short fiber aerogel scaffold obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the magnesium oxide-loaded asymmetric short fiber aerogel scaffold according to claim 8 in preparing a wound repair medical material.

10. Use of the magnesium oxide-loaded asymmetric short fiber aerogel scaffold according to claim 8 in preparing a medical material for promoting wound repair in diabetic patients.

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