Antibacterial bionic periosteum and its preparation method and application

Abstract

The present invention discloses an antibacterial bionic periosteum, a preparation method and application thereof, and relates to the field of biomedicine technology. The present invention obtains bioglass precursor fibers by adding phosphomolybdic acid to a bioglass precursor sol, and then performing electrostatic spinning, and finally heat-treating in a reducing atmosphere of ammonia to obtain a calcium molybdate / molybdenum nitride crystal phase, thereby obtaining an antibacterial bionic periosteum. The antibacterial bionic periosteum provided by the present invention has good biocompatibility, and has both multiple nanozyme properties and bone repair-promoting functions. On the one hand, its multiple nanozyme properties can generate ROS through multiple nanozyme properties, consume reducing substances, and inhibit the growth and reproduction of microorganisms such as bacteria; on the other hand, the antibacterial bionic periosteum can be used as a bioactive ceramic scaffold to promote the regeneration and repair of bone tissue cells.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an antibacterial bionic periosteum and a preparation method and application thereof. Background Art

[0002] Infected bone defects (IBD) are often caused by complex open fractures or orthopedic surgeries (such as radical tumor resection and implant placement). Severe cases of bone infection can result in amputation or even death. Bionic periosteum is an artificial material that mimics the structure and function of natural periosteum, designed to promote bone defect repair, accelerate bone regeneration, and integrate surrounding tissue. By integrating materials science and bioengineering, bionic periosteum attempts to replicate these properties, and has important applications in the repair of complex bone injuries, large bone defects, or after bone tumor resection.

[0003] Natural periosteum is flexible (it can bend and deform with bone), while most biomimetic materials in existing technologies (such as pure ceramics or hard polymers) are too rigid, easily creating a mechanical mismatch with surrounding tissues, leading to interfacial micromotion or stress shielding. Postoperative infection is a common complication of bone repair, but existing antibacterial designs of biomimetic periosteum are often accompanied by problems such as poor biocompatibility, cytotoxicity, and drug resistance. Furthermore, previous biomimetic periosteums have been functionally limited, failing to achieve both anti-infection and osteogenesis effects. To address these issues, the present invention proposes to develop a biomimetic periosteum with excellent biocompatibility to play a role in both anti-infection and osteogenesis. Summary of the Invention

[0004] The present invention aims to provide an antibacterial biomimetic periosteum and its preparation method and application to solve the problems existing in the above-mentioned prior art. The antibacterial biomimetic periosteum has good biocompatibility and has both anti-infection and bone repair functions.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for preparing an antibacterial bionic periosteum, comprising the following steps:

[0007] Solution B is added to solution A to obtain a sol solution through a mixing reaction, and then a polyvinyl butyral-ethanol solution is added to obtain a mixed spinning solution. After stirring to evaporate the solvent, electrospinning is performed to obtain a bioactive glass membrane precursor.

[0008] The bioactive glass membrane precursor is dried to volatilize the residual solvent, and then subjected to heat treatment in a reducing atmosphere of ammonia to obtain the antibacterial biomimetic periosteum;

[0009] The preparation method of the solution A comprises: dissolving tetraethoxysilane and triethyl phosphate in a solvent, and then adding Ca(NO3)2·4H2O to obtain the solution A;

[0010] The preparation method of the solution B comprises: adding deionized water and calcium carbonate to a phosphomolybdic acid solution, and fully dissolving the calcium carbonate to obtain the solution B.

[0011] Furthermore, the concentration of the polyvinyl butyral-ethanol solution is 6% w / v.

[0012] Furthermore, the solvent is ethanol.

[0013] Furthermore, the temperature of the drying treatment is 25-100°C.

[0014] Furthermore, the heat treatment temperature is 600-700° C. and the time is 0.5-1 h.

[0015] Furthermore, the ratio of the tetraethoxysilane, the triethyl phosphate and the Ca(NO3)2·4H2O is (14-16) mL: (0.5-1) mL: (3-4) g.

[0016] Furthermore, the ratio of the phosphomolybdic acid solution, the deionized water and the calcium carbonate is (3-4) mL: (1-2) mL: (0.01-0.05) g.

[0017] The present invention also provides an antibacterial bionic periosteum prepared according to the above preparation method.

[0018] The present invention also provides application of the antibacterial bionic periosteum in preparing surgical products for treating infected bone defects.

[0019] The present invention also provides a surgical product for preparing and treating infected bone defects, wherein the active component includes the above-mentioned antibacterial bionic periosteum.

[0020] The present invention discloses the following technical effects:

[0021] The present invention develops an antibacterial bionic periosteum, which is prepared by adding phosphomolybdic acid to a bioglass precursor sol, then performing electrospinning to obtain bioglass precursor fibers, and finally heat-treating them in a reducing atmosphere of ammonia to obtain a calcium molybdate / molybdenum nitride crystal phase, thereby obtaining the antibacterial bionic periosteum.

[0022] The antibacterial biomimetic periosteum provided by this invention exhibits excellent biocompatibility and combines multiple nanozyme properties with bone repair capabilities. On the one hand, these nanozyme properties, such as peroxidase (POD), glutathione oxidase (GSHOx), and catalase (CAT), generate ROS, consuming reducing substances to inhibit the growth and reproduction of bacteria and other microorganisms. On the other hand, the antibacterial biomimetic periosteum can serve as a bioactive ceramic scaffold to promote the regeneration and repair of bone tissue cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 The following are microscopic images of three biomimetic periosteums under scanning electron microscope (SEM);

[0025] Figure 2 Energy dispersive X-ray spectra of CaMoO4 / Mo3N2-BGFM;

[0026] Figure 3 X-ray diffraction (XRD) patterns of CaMoO4, Mo3N2 and CaMoO4 / Mo3N2;

[0027] Figure 4 Figure 2 is the detection result of POD enzyme activity;

[0028] Figure 5 Figure 1 is a graph showing the biocompatibility test results of the antibacterial bionic periosteum; Figure a is a scanning electron microscope observation of the adhesion state of bone marrow mesenchymal stem cells and the antibacterial bionic periosteum; Figure b is a graph showing the cell activity and toxicity test results;

[0029] Figure 6 The results of detecting the expression of COL1A1 in biomimetic periosteum by cell immunofluorescence technology; a is the cell immunofluorescence detection diagram; b is the statistical diagram of relative fluorescence intensity;

[0030] Figure 7 The results of detecting the expression of BMP2 in biomimetic periosteum by cell immunofluorescence technology; a is the cell immunofluorescence detection graph; b is the statistical graph of relative fluorescence intensity;

[0031] Figure 8 Alkaline phosphatase staining images of different biomimetic periosteum (a) and statistical graphs of integrated optical density (b);

[0032] Figure 9 Alizarin Red S staining images of different biomimetic periosteum (a) and statistical graph of absorbance at 60 nm (b);

[0033] Figure 10 ROS flow cytometry detection results (a) and statistical graph of mean fluorescence intensity (b) of different biomimetic periostea;

[0034] Figure 11 The results of the in vitro antibacterial function test of the bionic periosteum are shown in Figure 1. a is the bacterial live / dead staining result; b is the scanning electron microscope observation image; c is the ROS staining image; NIR represents the photothermal reaction;

[0035] Figure 12 Figure 1 shows the micro-computed tomography (Micro CT) results of different bionic periosteums; the scale is 2 mm.

[0036] Figure 13 H&E staining images of different biomimetic periosteum;

[0037] Figure 14 The immunohistochemical staining images of different biomimetic periosteums;

[0038] Figure 15 Figure 2 is the body temperature monitoring results of rats in each experimental group;

[0039] Figure 16 Figure 2 is the blood routine test results of rats in each experimental group;

[0040] Figure 17 Wright-Giemsa staining images of rats in each experimental group; NIR represents photothermal reaction;

[0041] Figure 18 are the immunohistochemical staining images of rats in each experimental group; NIR represents photothermal reaction;

[0042] Figure 19 The diagram shows the tissue colony detection results of the femoral bone marrow of rats in each experimental group; NIR stands for photothermal reaction. DETAILED DESCRIPTION

[0043] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0044] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0045] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0046] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0047] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0048] The antibacterial biomimetic periosteum developed by the present invention is a bioglass fiber membrane. It is prepared by adding phosphomolybdic acid to a bioglass precursor sol, followed by electrospinning to obtain bioglass precursor fibers. Finally, heat treatment in a reducing atmosphere of ammonia produces a calcium molybdate / molybdenum nitride crystalline phase. Because the spinning solution used in the present invention is a high-concentration bioglass sol, the choice of molybdenum source is very critical. The present invention selected phosphomolybdic acid, a molybdenum source that is easily soluble in ethanol, for experiments and found that it has good compatibility with the bioglass sol system. Therefore, it is possible to successfully prepare a bioglass sol containing a highly active molybdenum source, and then spin it through electrospinning technology to obtain a bioactive glass membrane precursor containing a highly active molybdenum source. Subsequently, the present invention achieves the formation of calcium molybdate in the biomimetic periosteum through heat treatment and a reducing atmosphere medium, on the one hand, and the formation of molybdenum nitride therein, on the other hand, to ultimately obtain a calcium molybdate / molybdenum nitride-biomimetic periosteum. The simultaneous generation of these two forms a heterogeneous structure that can generate efficient electron transfer, achieving effective catalytic cracking of hydrogen peroxide molecules and generating ROS.

[0049] Example 1

[0050] Preparation of biomimetic periosteum (CaMoO4 / Mo3N2-BGFM):

[0051] The present invention utilizes the principle of linear aggregates generated by partial hydrolysis of alkoxysilane, and prepares biomimetic periosteum by a sol-gel method with the addition of phosphomolybdic acid and an electrospinning process. The specific steps are as follows:

[0052] Prepare solution A: Dissolve 15 mL of tetraethoxysilane (TEOS) and 0.705 mL of triethyl phosphate in 7 mL of ethanol, stir, and then add 3.2 g of Ca(NO3)2·4H2O.

[0053] Prepare solution B: Add 1.45 mL of deionized water to 3.65 mL of a 20 wt% phosphomolybdic acid ethanol solution, mix well, then add 0.03 g of calcium carbonate (CaCO3). Ultrasonicate for 30 minutes to ensure that the CaCO3 is fully dissolved.

[0054] Solution B was slowly added dropwise to solution A and stirred for 2 hours to obtain a sol. Next, 2 mL of a 6% w / v polyvinyl butyral (PVB)-ethanol solution was added to the sol to obtain a mixed spinning solution to induce phase separation for electrospinning fibers. The resulting mixed spinning solution was stirred for 36 hours, with the solvent continuously evaporated to increase the viscosity of the sol. When the total liquid volume was reduced to 20 mL, the aged mixed spinning solution was loaded into a plastic syringe equipped with a metal needle (21-gauge needle, 0.5 mm diameter). A high-voltage electric field of 15 kV was applied to the needle and collection plate to perform electrospinning. The electrospun bioactive glass membrane precursor was then transferred to a 50°C oven and baked for 12 hours to evaporate any residual solvent. The biomimetic periosteum containing CaMoO₄ and Mo₃N₂ was then heat-treated by heating to 700°C in an ammonia atmosphere and holding for 1 hour to obtain the biomimetic periosteum.

[0055] Example 2

[0056] 1. Preparation of biomimetic periosteum

[0057] Preparation of biomimetic periosteum (CaMoO4 / Mo3N2-BGFM):

[0058] The present invention utilizes the principle of linear aggregates generated by partial hydrolysis of alkoxysilane, and prepares biomimetic periosteum by a sol-gel method with the addition of phosphomolybdic acid and an electrospinning process. The specific steps are as follows:

[0059] Prepare solution A: Dissolve 14 mL of TEOS and 0.5 mL of triethyl phosphate in 7 mL of ethanol, stir, and then add 3 g of Ca(NO3)2·4H2O.

[0060] Prepare solution B: Add 2 mL of deionized water to 3 mL of 20 wt% phosphomolybdic acid ethanol solution, mix well, then add 0.01 g of CaCO3 and sonicate for 30 minutes to ensure that the CaCO3 is fully dissolved.

[0061] Solution B was slowly added dropwise to solution A and stirred for 2 hours to obtain a sol. Next, 2 mL of a 6% w / v polyvinyl butyral (PVB)-ethanol solution was added to the sol to obtain a mixed spinning solution to induce phase separation for electrospinning fibers. The resulting mixed spinning solution was stirred for 36 hours, with the solvent continuously evaporated to increase the viscosity of the sol. When the total liquid volume was reduced to 20 mL, the aged mixed spinning solution was loaded into a plastic syringe equipped with a metal needle (21-gauge needle, 0.5 mm diameter). A high-voltage electric field of 15 kV was applied to the needle and collection plate to perform electrospinning. The electrospun bioactive glass membrane precursor was then transferred to a 25°C oven and baked for 12 hours to evaporate any residual solvent. The biomimetic periosteum containing CaMoO₄ and Mo₃N₂ was then heat-treated by heating to 600°C in an ammonia atmosphere for 0.8 h.

[0062] Example 3

[0063] Preparation of biomimetic periosteum (CaMoO4 / Mo3N2-BGFM):

[0064] The present invention utilizes the principle of linear aggregates generated by partial hydrolysis of alkoxysilane, and prepares biomimetic periosteum by a sol-gel method with the addition of phosphomolybdic acid and an electrospinning process. The specific steps are as follows:

[0065] Prepare solution A: Dissolve 16 mL of TEOS and 0.5 mL of triethyl phosphate in 7 mL of ethanol, stir, and then add 4 g of Ca(NO3)2·4H2O.

[0066] Prepare solution B: Add 1 mL of deionized water to 4 mL of 20 wt% phosphomolybdic acid ethanol solution, mix well, then add 0.05 g of CaCO3 and sonicate for 30 minutes to ensure that the CaCO3 is fully dissolved.

[0067] Solution B was slowly added dropwise to solution A and stirred for 2 hours to obtain a sol. Next, 2 mL of a 6% w / v polyvinyl butyral (PVB)-ethanol solution was added to the sol to obtain a mixed spinning solution to induce phase separation for electrospinning fibers. The resulting mixed spinning solution was stirred for 36 hours, with the solvent continuously evaporated to increase the viscosity of the sol. When the total liquid volume was reduced to 20 mL, the aged mixed spinning solution was loaded into a plastic syringe equipped with a metal needle (21-gauge needle, 0.5 mm diameter). A high-voltage electric field of 15 kV was applied to the needle and collection plate to perform electrospinning. The electrospun bioactive glass membrane precursor was then transferred to an oven at 100°C (the temperature could be controlled between 25 and 100°C) for 12 hours to evaporate any residual solvent. The biomimetic periosteum containing CaMoO₄ and Mo₃N₂ was then heat-treated by heating to 650°C in an ammonia atmosphere and holding for 0.5 h.

[0068] Example 4

[0069] 1. Experimental Methods

[0070] The following combines calcium molybdate bionic periosteum (CaMoO4-BGFM) and molybdenum nitride bionic periosteum (Mo3N2-BGFM), taking the bionic periosteum prepared in Example 1 as an example to characterize and illustrate its performance.

[0071] The preparation method of calcium molybdate biomimetic periosteum (CaMoO4-BGFM) is the same as that of Example 1, except that the heat treatment is to heat to 800°C in an air atmosphere and keep the temperature for 2 hours.

[0072] The preparation method of molybdenum nitride biomimetic periosteum (Mo3N2-BGFM) is the same as that of Example 1, except that the heat treatment is to heat to 800°C in an ammonia atmosphere and keep the temperature for 2 hours.

[0073] 1. Characterization of the biomimetic periosteum

[0074] The morphology and elemental composition of the antibacterial biomimetic periosteum were investigated using field scanning electron microscopy and energy dispersive X-ray spectroscopy (EDS). The phase composition was measured by X-ray diffraction (XRD).

[0075] The ability of different biomaterials (CaMoO4, Mo3N2, CaMoO4+Mo3N2, and CaMoO4 / Mo3N2) to react with peroxidase (POD) that catalyzes H2O2 was tested as follows: 10 μL of biomaterial dispersion (CaMoO4, Mo3N2, or CaMoO4 / Mo3N2, all at a concentration of 20 mg / mL) and 40 μL of H2O2 (100 mM) were added to 1.95 mL of acetic acid-sodium acetate buffer (0.1 M, pH = 5.0). For the CaMoO4+Mo3N2 group, 10 μL each of CaMoO4 dispersion (20 mg / mL) and Mo3N2 dispersion (20 mg / mL) were added to the reaction system (the buffer was reduced to ensure a total reaction system of 2 mL). Subsequently, 100 μL of 3,3',5,5'-tetramethylbiphenyl (TMB, 20 mM) was added to the above mixture. A color reaction was generated by POD, and the absorbance within the wavelength range of 400-800 nm was recorded using UV-visible spectrophotometry to measure the amount of TMB oxidation product (oxTMB). A blank control (CN) was also set up, in which an equal amount of blank solvent was used instead of the biomaterial dispersion.

[0076] 2. Biocompatibility of antibacterial biomimetic periosteum

[0077] 2.1 Scanning electron microscopy

[0078] Bone marrow mesenchymal stem cells (BMMSCs) were seeded on different biomimetic periosteum surfaces and then fixed, dehydrated, and gold-sprayed. The adhesion state of BMMSCs to the biomimetic periosteum was observed using a scanning electron microscope.

[0079] 2.2 Cell viability and toxicity assays

[0080] Cells were stained using a live / dead staining kit (Beyotime, China). BMMSCs were seeded on different antibacterial biomimetic periosteum surfaces and cultured for 48 hours. Calcein AM / PI working solution was added to the cell culture dishes and incubated at 37°C for 30 minutes. Cell fluorescence was observed under an inverted fluorescence microscope.

[0081] 3. In vitro and in vivo osteogenesis tests

[0082] 3.1 ROS level detection

[0083] After culturing BMMSCs on different biomaterials, the cells were collected and stained using a ROS detection kit (Beyotime, China). The cellular ROS levels were detected using a flow cytometer (BD, USA), and the results were analyzed using FlowJo_V10.

[0084] 3.2 Immunofluorescence detection

[0085] After culturing BMMSCs on various biomaterials, the cells were fixed with 4% paraformaldehyde, washed three times with PBS, and permeabilized with 0.5% Triton X-100 for 15 minutes. After blocking with 20% goat serum, the cells were incubated with the primary antibody overnight at 4°C. After incubation with fluorescent secondary antibodies, the cytoskeleton and nuclei were stained with phalloidin and DAPI, respectively, and the cells were immediately observed under an inverted fluorescence microscope.

[0086] 3.3 Alkaline phosphatase (ALP) staining

[0087] After 7 days of continuous osteogenic induction, ALP levels were measured using an alkaline phosphatase staining kit (Beyotime, China). After fixation, the cells were added to the cell culture dish with the staining solution. After 1 hour of reaction, the reaction was terminated with deionized water and observed under an inverted microscope.

[0088] 3.4 Alizarin Red S (ARS) staining

[0089] After 21 days of continuous osteogenic induction, the cells were fixed and stained with Alizarin Red S stain (Solabo, China) for 1 hour. The excess dye was washed off with PBS and the cells were observed under an inverted microscope.

[0090] 3.5 Establishment of rat femoral defect model

[0091] Rats were anesthetized with sodium pentobarbital, and the hair at the surgical site was removed. After disinfection, the muscles were separated layer by layer using sterile scissors until the femur was observed. The periosteum on the surface of the femur was cut off, and a single cortical bone defect with a diameter of 4 mm was made above the intercondylar space of the femur using a sterile trephine drill.

[0092] 3.6 Micro-CT detection

[0093] At four and eight weeks after successful bone defect model establishment, the rats were euthanized, and the femurs were carefully removed and fixed in 4% paraformaldehyde for 24 hours. The tissues were scanned using a high-resolution micro-CT scanner (Perkin Elmer, Japan). Parameters were adjusted to 90 kV, 88 μA, and a resolution of 7 μm per pixel. Images were reconstructed using CTAn software (Perkin Elmer, Japan), and sample parameters were analyzed using CTVox software (Perkin Elmer, Japan).

[0094] 3.7 Histological staining

[0095] At eight weeks postoperatively, rats were euthanized, and femurs were isolated and fixed in 4% paraformaldehyde for 24 hours. Samples were then decalcified with ethylenediaminetetraacetic acid (EDTA) for two weeks. The specimens were embedded in paraffin and cut into 5 μm sections. Sections were stained using the H&E staining kit (Solebro, China) and the Masson staining kit (Solebro, China) and observed under an inverted microscope.

[0096] 3.8 Immunohistochemical staining

[0097] For immunohistochemical staining, deparaffinized sections were treated with 3% H₂O₂ for 5 minutes and then with 5% BSA for 10 minutes. Sections were then incubated with primary antibodies overnight at 4°C. Subsequently, they were incubated with biotin-conjugated secondary antibodies and visualized using streptavidin-biotin staining. Cell nuclei were stained with hematoxylin, and slides were photographed using a microscope.

[0098] 4 In vitro and in vivo antibacterial tests

[0099] 4.1 Establishment of rat femoral defect infection model

[0100] After anesthetizing rats with sodium pentobarbital, the hair at the surgical site was removed. After disinfection, sterile scissors were used to separate the muscles layer by layer until the femur was visible. The periosteum on the surface of the femur was sheared off, and a sterile trephine was used to create a 4mm diameter monocortical bone defect above the intercondylar space of the femur. To establish a bone defect infection model, the present invention filled the bone defect with gelatin sponge soaked in a suspension of methicillin-resistant Staphylococcus aureus (MASR). The experimental groups covered the bone defect with different biomimetic periosteums (CaMoO4, MO3N2, or CaMoO4 / MO3N2), while the control group did not receive any covering treatment. Each group was sutured layer by layer. MO3N2+NIR and CaMoO4 / MO3N2+NIR groups were also established. In addition to covering the biomimetic periosteum, photothermal reaction treatment was added, with a laser power of 0.8W / cm 2 , the treatment time is 10 minutes.

[0101] 4.2 General Condition and Hematological Analysis of Animals

[0102] Before establishing the osteomyelitis model, the initial body weight and body temperature of each group of rats were measured. Body temperature was then routinely monitored daily for 2 weeks after treatment. Venous blood was obtained from the rats on days 3, 7, and 14 after treatment. Standard blood tests were performed to assess the level of systemic infection in these rats.

[0103] 4.3 Tissue colony plate test

[0104] After 3 days of treatment of the infection model, the wound was opened again and bone marrow was obtained from the femur. The bone marrow was immediately placed in 1 mL of sterile saline and shaken evenly. After dilution, the bone marrow was spread on LB agarose gel and incubated at 37°C for 12 hours before the CFU value was calculated.

[0105] 4.4 H&E staining and Giemsa staining

[0106] After 14 days of treatment, rats were euthanized, and femurs were removed, fixed with 4% paraformaldehyde, decalcified with EDTA, and then sectioned. Sections were stained using a Giemsa staining kit (Solarbio, China) and observed under an inverted microscope.

[0107] 5. Statistical Analysis

[0108] All data are presented as mean ± standard deviation (SD). All experiments were repeated at least three times. Comparisons between two groups were performed using the Student's t-test, and comparisons between multiple groups were performed using one-way analysis of variance (ANOVA). Statistical significance was set at P < 0.05.

[0109] 2. Experimental Results

[0110] 1. Characterization of the Antibacterial Bionic Periosteum

[0111] like Figure 1 As shown in the figure, all three bionic periosteums showed good continuity, the fiber diameter was about 800-1000 nm, and the fiber interior showed porous characteristics. This microstructure is conducive to the degradation of the fiber. It can be speculated that the heterojunction nanozymes produced by the degradation can be swallowed by bacteria, produce multiple catalytic effects to generate ROS, and inhibit the growth and reproduction of bacteria.

[0112] like Figure 2 As shown, the elements of CaMoO4 / Mo3N2-BGFM fibers are evenly distributed, which proves the uniformity of the spinning solution sol. The resulting heterojunction nanozymes are evenly distributed in the fibers, which is beneficial to improving their catalytic activity.

[0113] like Figure 3 As shown, the three biomimetic periosteums have the physical phases of CaMoO4, Mo3N2 and CaMoO4 / Mo3N2, respectively, proving the successful preparation of bioglass fibers with CaMoO4 / Mo3N2 heterogeneous structure.

[0114] 2. POD enzyme activity test results

[0115] The results of POD enzyme activity test ( Figure 4) It can be found that the CaMoO4 / Mo3N2-BGFM group has extremely excellent POD enzyme activity. Compared with single-component biofiber materials (CaMoO4 and Mo3N2) or a simple blend of two single-component fibers (CaMoO4+Mo3N2), its POD activity has achieved a qualitative leap, which is attributed to the formation of heterogeneous structures in the fibers.

[0116] 3. Biocompatibility test results of biomimetic periosteum

[0117] Scanning electron microscopy revealed a continuous contact area between the cells and the material surface, indicating good adhesion. Furthermore, cells were observed to extend pseudopodia and actively contact the material, indicating that cells were able to carry out normal physiological activities on the material surface. Figure 5 (a) Cell live / dead staining results showed that the vast majority of BMMSCs showed green fluorescence (Calcein-AM staining), indicating a high cell survival rate. The number of dead cells (red fluorescence, PI staining) was very small and sparsely distributed, indicating that the material had low cytotoxicity ( Figure 5 Middle b).

[0118] 4. In vitro osteogenic function test results of biomimetic periosteum

[0119] In order to more intuitively observe the expression differences of osteogenesis-related genes COL1A1 and BMP2, the present invention detected the expression levels of COL1A1 and BMP2 by cell immunofluorescence technology. The CaMoO4 / MO3N2 group had the highest average fluorescence intensity ( Figure 6-Figure 7 ); Alkaline phosphatase staining and Alizarin Red S staining also observed a higher positive rate and calcium salt deposition ( Figure 8-Figure 9 ); Flow cytometry showed that the antibacterial biomimetic periosteum could significantly alleviate the ROS level in bone marrow mesenchymal stem cells ( Figure 10 These results indicate that CaMoO4 / MO3N2 biomimetic periosteum can effectively reduce cellular ROS levels and promote osteogenic differentiation.

[0120] 5. In vitro antibacterial function test results of antibacterial biomimetic periosteum

[0121] The results of bacterial live / dead staining showed that compared with the control group (Control), the average survival rates of MASR in the CaMoO4 group, MO3N2 group, MO3N2+NIR group, CaMoO4 / MO3N2 group, and CaMoO4 / MO3N2+NIR group were 67.91%, 40.77%, 21.71%, 36.51%, and 16.52%, respectively, all lower than the 97.84% survival rate of the control group. The survival rates of E. coli were 81.05%, 81.39%, 51.93%, 57.29%, and 41.41%, respectively, also lower than the 97.84% survival rate of the control group ( Figure 11 (a) Scanning electron microscopy results showed that the MASR in the control group showed a typical spherical shape, while E. coli showed a typical rod shape, and both bacteria were plump. However, after co-culture with the biomimetic periosteum, MASR and E. coli lost their typical morphological characteristics, with the bacteria deforming or shrinking, and the surface becoming concave or even perforated. The bacterial state of the CaMoO4 / MO3N2 group was worse than that of the CaMoO4 and MO3N2 groups, and a stronger antibacterial effect was obtained through photothermal reaction (CaMoO4 / MO3N2+NIR group) ( Figure 11 Middle b); ROS staining results showed that the ROS level of bacteria co-cultured with the antibacterial biomimetic periosteum was higher than that of the control group, and this effect was most significant in the CaMoO4 / MO3N2+NIR group ( Figure 11 These results indicate that the CaMoO4 / MO3N2 biomimetic periosteum has good antibacterial function in vitro.

[0122] 6. In vivo osteogenic function test results of antibacterial biomimetic periosteum

[0123] Micro CT scan results showed that at the 4th week of bone defect healing, sunken cortical bone and hyperplastic bone could still be observed at the bone defect sites of each group, but the implantation of antibacterial bionic periosteum significantly promoted bone defect repair. At the 8th week, obvious bone defects could still be observed in the control group (Control), and the degree of bone defect repair in the CaMoO4 and MO3N2 groups was significantly better than that in the control group. The bone defect surface of the CaMoO4 / MO3N2 group was significantly smoother than that of the control group, and had higher cortical bone thickness (Ct.Th), cortical bone area (Ct.Ar), and cortical bone area ratio (Ct.Ar / Tt.Ar) than other groups. Figure 12 H&E staining results showed that at week 8, the cortical bone in the control group was sparse and disordered, while the cortical bone in the CaMoO4 and MO3N2 groups was significantly more abundant and orderly than that in the control group. The cortical bone in the CaMoO4 / MO3N2 group was uniform and dense ( Figure 13 OPN and BMP2 are key regulators of bone matrix formation, mineralization, and bone remodeling. Immunohistochemical staining results showed that the most positive foci were observed in the CaMoO4 / MO3N2 group, indicating that bone repair mediated by osteogenic differentiation of BMMSCs at the bone defect site in the CaMoO4 / MO3N2 group was more active ( Figure 14 These results indicate that CaMoO4 / MO3N2 biomimetic periosteum can promote bone defect repair in vivo.

[0124] 7. In vivo antibacterial function test results of antibacterial biomimetic periosteum

[0125] like Figure 15As shown, the present invention monitored the body temperature changes of rats within 2 weeks. The results showed that the body temperature of the rats was within the normal range before surgery, reached a peak on the first day after surgery, and there was no difference between the groups. Subsequent body temperature records showed that compared with the control group (Control), the experimental group using bionic periosteum recovered body temperature faster. On the 10th day after surgery, the rats in the CaMoO4 / MO3N2 group recovered to their preoperative body temperature. The control group did not recover their body temperature until the 14th day after surgery, and the recovery level of the CaMoO4 / MO3N2 group only reached the recovery level of the CaMoO4 / MO3N2 group on the 9th day. Subsequently, the present invention monitored the changes in the rats' venous blood routine. The results showed that on the first day after surgery, the white blood cells (WBC) of each group exceeded the normal value, among which the WBC index of the control group was the highest, reaching 2.08×10 10 The main symptom was an increase in granulocytes. The results of routine blood tests on the 7th, 14th, and 21st days after surgery showed that implanting the bionic periosteum could significantly alleviate the abnormalities in blood routine caused by infection. This effect was most obvious in the CaMoO4 / MO3N2 group ( Figure 16 ). The present invention then evaluated the degree of femoral infection in rats by Wright-Giemsa staining and immunohistochemical staining. The results of Wright-Giemsa staining showed that the number of Giemsa-positive foci was significantly reduced after implantation of the bionic periosteum, with the CaMoO4 / MO3N2 group showing the most significant effect ( Figure 17 ). Subsequently, in order to further evaluate the activity of neutrophils in the bone marrow cavity, the present invention performed myeloperoxidase (MPO) immunohistochemical staining on the tissue sections. The results showed that the number of MPO-positive cells in the bone marrow of the control group increased significantly, indicating a large infiltration of neutrophils and a strong inflammatory response. The number of MPO-positive cells in the rats implanted with biomimetic periosteum decreased significantly, and the distribution range was narrowed, indicating that the material effectively inhibited neutrophil infiltration and inflammatory response ( Figure 18 ).

[0126] Finally, the present invention obtained bone marrow from a rat femoral defect infection model, diluted it, and inoculated it on agarose gel for culture. The results showed that the MASR growth density in the control group (Control) was the highest, and the MASR growth density in the CaMoO4 / MO3N2+NIR group was effectively controlled ( Figure 19 ).

[0127] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing an antibacterial bionic periosteum, characterized in that: The following steps are involved: Solution B is added to solution A to obtain a sol solution through a mixing reaction, and then a polyvinyl butyral-ethanol solution is added to obtain a mixed spinning solution. After stirring to evaporate the solvent, electrospinning is performed to obtain a bioactive glass membrane precursor. The bioactive glass membrane precursor is dried to volatilize the residual solvent, and then subjected to heat treatment in a reducing atmosphere of ammonia to obtain the antibacterial biomimetic periosteum; The preparation method of the solution A comprises: dissolving tetraethoxysilane and triethyl phosphate in a solvent, and then adding Ca(NO3)2·4H2O to obtain the solution A; The preparation method of the solution B comprises: adding deionized water and calcium carbonate to a phosphomolybdic acid solution and fully dissolving the calcium carbonate to obtain the solution B; The concentration of the polyvinyl butyral-ethanol solution is 6% w / v; The heat treatment temperature is 600-700°C and the time is 0.5-1h; The ratio of the tetraethoxysilane, the triethyl phosphate and the Ca(NO3)2·4H2O is (14-16) mL: (0.5-1) mL: (3-4) g; The ratio of the phosphomolybdic acid solution, the deionized water and the calcium carbonate is (3-4) mL: (1-2) mL: (0.01-0.05) g.

2. The preparation method according to claim 1, characterized in that The solvent is ethanol.

3. The preparation method according to claim 1, characterized in that The temperature of the drying treatment is 25-100°C.

4. An antibacterial bionic periosteum prepared according to the preparation method according to any one of claims 1 to 3.

5. Use of the antibacterial bionic periosteum according to claim 4 in preparing surgical products for treating infected bone defects.

6. A surgical product for treating infected bone defects, characterized in that: The active ingredient comprises the antibacterial biomimetic periosteum according to claim 4.

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