Antibacterial bionic periosteum and preparation method and application thereof

Abstract

The invention discloses an antibacterial bionic periosteum as well as a preparation method and application thereof, and relates to the technical field of biological medicines. The preparation method comprises the following steps: adding phosphomolybdic acid into bioglass precursor sol, then carrying out electrostatic spinning to obtain bioglass precursor fibers, and finally carrying out heat treatment in a reducing atmosphere of ammonia gas to obtain a calcium molybdate / molybdenum nitride crystal phase, thereby obtaining the antibacterial bionic periosteum. The antibacterial bionic periosteum provided by the invention has good biocompatibility, and has the characteristics of various nano enzymes and the function of promoting bone repair. On one hand, ROS can be generated through multiple nano-enzyme properties, reducing substances are consumed, and growth and reproduction of microorganisms such as bacteria are inhibited; on the other hand, the antibacterial bionic periosteum can be used as a bioactive ceramic scaffold to promote regeneration and repair of bone tissue cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to an antibacterial bionic periosteum and its preparation method and application. Background Art

[0002] Infectious bone defect (IBD) is often caused by complex open fractures or orthopedic surgeries (such as radical tumor resection, implants). Cases of severe bone infection face the risk of amputation or even death. Bionic Periosteum is an artificial material that mimics the structure and function of natural periosteum, aiming to promote bone defect repair, accelerate bone regeneration, and integrate surrounding tissues. Through the combination of materials science and bioengineering, bionic periosteum attempts to reproduce these characteristics, and has important applications especially in the repair of complex bone injuries, large-area bone defects, or after bone tumor resection.

[0003] Natural periosteum has flexibility (it can bend and deform with the bone), while most bionic materials in the prior art (such as pure ceramics or rigid polymers) are too rigid, which easily causes mechanical mismatch with surrounding tissues, resulting in interfacial micro-motion or stress shielding. Postoperative infection is a common complication in bone repair, but the antibacterial design of existing bionic periosteum often has problems such as poor biocompatibility, cytotoxicity, and drug resistance. And previous bionic periosteum showed a single function in the past and could not have the dual effects of anti-infection and bone formation. Based on the above problems, the present invention intends to develop a bionic periosteum with good biocompatibility, in order to play a role in anti-infection and bone formation. Summary of the Invention

[0004] The purpose of the present invention is to provide an antibacterial bionic periosteum and its preparation method and application to solve the problems existing in the above prior art. This antibacterial bionic periosteum has good biocompatibility and combines the functions of anti-infection and promoting bone repair.

[0005] To achieve the above purpose, the present invention provides the following solution:

[0006] The present invention provides a preparation method of an antibacterial bionic periosteum, including the following steps:

[0007] Add solution B to solution A, obtain a sol solution through mixing reaction, then add polyvinyl butyral-ethanol solution to obtain a mixed spinning solution, stir to volatilize the solvent and then perform electrospinning to obtain a bioactive glass membrane precursor;

[0008] Perform drying treatment on the bioactive glass membrane precursor, volatilize the residual solvent, and then perform heat treatment in a reducing atmosphere of ammonia gas to obtain the antibacterial bionic periosteum;

[0009] The preparation method of the solution A includes: 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 includes: adding deionized water and calcium carbonate to a phosphomolybdic acid solution, and after fully dissolving, the solution B is obtained.

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

[0012] Further, the solvent is ethanol.

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

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

[0015] Further, 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] Further, 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 biomimetic bone membrane prepared by the above preparation method.

[0018] The present invention also provides the application of the above antibacterial biomimetic bone membrane in the preparation of surgical supplies for treating infectious bone defects.

[0019] The present invention also provides a method for preparing surgical supplies for treating infectious bone defects, and the active ingredient includes the above antibacterial biomimetic bone membrane.

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

[0021] The present invention develops an antibacterial biomimetic bone membrane. By adding phosphomolybdic acid to a bioactive glass precursor sol, bioactive glass precursor fibers are obtained through electrospinning, and finally, calcium molybdate / molybdenum nitride crystal phases are obtained by heat treatment in a reducing atmosphere of ammonia, thereby obtaining the antibacterial biomimetic bone membrane.

[0022] The antibacterial bionic periosteum provided by the present invention has good biocompatibility, and also has a variety of nanozyme characteristics and osteogenic repair-promoting functions. On the one hand, its various nanozyme characteristics can generate ROS through the properties of various nanozymes such as peroxidase (POD), glutathione oxidase (GSHOx), and catalase (CAT), consume reducing substances, and inhibit the growth and reproduction of bacteria and other microorganisms; on the other hand, this antibacterial bionic periosteum can be used as a bioactive ceramic scaffold to promote the regeneration and repair of bone tissue cells. Description of the Drawings

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0024] Figure 1 are the microscopic images of three bionic periosteums under a scanning electron microscope (SEM);

[0025] Figure 2 is the energy-dispersive X-ray spectroscopy diagram of CaMoO4 / Mo3N2-BGFM;

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

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

[0028] Figure 5 are the detection result diagrams of the biocompatibility of the antibacterial bionic periosteum; among them, a is the scanning electron microscope observation diagram of the adhesion state of bone marrow mesenchymal stem cells and the antibacterial bionic periosteum; b is the detection result diagram of cell activity and toxicity;

[0029] Figure 6 are the results of detecting the expression level of COL1A1 of the bionic periosteum by cell immunofluorescence technology; among them, a is the cell immunofluorescence detection diagram; b is the statistical chart of relative fluorescence intensity;

[0030] Figure 7 are the results of detecting the expression level of BMP2 of the bionic periosteum by cell immunofluorescence technology; among them, a is the cell immunofluorescence detection diagram; b is the statistical chart of relative fluorescence intensity;

[0031] Figure 8 are the alkaline phosphatase staining diagrams (a) and the statistical charts of integrated optical density (b) of different bionic periosteums;

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

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

[0034] Figure 11 Detection result images of the in vitro antibacterial function of the biomimetic periosteum; among them, a is the live / dead bacteria staining result; b is the observation image by scanning electron microscope; c is the ROS staining image; NIR represents the photothermal reaction;

[0035] Figure 12 Micro-computed tomography (Micro CT) result images of different biomimetic periosteums; the scale bar is 2 mm for all;

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

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

[0038] Figure 15 Body temperature monitoring result images of rats in each experimental group;

[0039] Figure 16 Blood routine test result images of rats in each experimental group;

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

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

[0042] Figure 19 Tissue colony detection result images of the femoral bone marrow of rats in each experimental group; NIR represents the photothermal reaction. Detailed implementation manners

[0043] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation to the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0044] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0045] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0046] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0047] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0048] The antibacterial bionic periosteum developed in the present invention is a bioactive glass fiber membrane, which is obtained by adding phosphomolybdic acid to a bioactive glass precursor sol, then performing electrospinning to obtain bioactive glass precursor fibers, and finally heat-treating in a reducing atmosphere of ammonia gas to obtain a calcium molybdate / molybdenum nitride crystal phase. Since the spinning solution used in the present invention is a high-concentration bioactive glass sol, the selection of the molybdenum source is very demanding. In the present invention, phosphomolybdic acid, a molybdenum source that is easily soluble in ethanol, is selected for experiments and it is found that it has good compatibility with the bioactive glass sol system. Therefore, a bioactive glass sol containing a highly active molybdenum source can be successfully prepared, and then a bioactive glass membrane precursor containing a highly active molybdenum source can be obtained by electrospinning technology. After that, through heat treatment and a reducing atmosphere medium, on the one hand, the formation of calcium molybdate in the bionic periosteum is achieved, and on the other hand, the formation of molybdenum nitride therein is achieved, and finally a calcium molybdate / molybdenum nitride - bionic periosteum is obtained. The heterostructure formed by the simultaneous generation of the two can produce effective electron transfer and realize the effective catalytic cleavage of hydrogen peroxide molecules to produce ROS.

[0049] Example 1

[0050] Preparation of Bionic Periosteum (CaMoO4 / Mo3N2-BGFM):

[0051] Based on the principle that partial hydrolysis of alkoxysilane produces linear aggregates, the bionic periosteum is prepared by the sol-gel method with the addition of phosphomolybdic acid and the electrospinning process. The specific steps are as follows:

[0052] Prepare Solution A: Dissolve 15 mL of tetraethyl orthosilicate (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% ethanol solution of phosphomolybdic acid, mix well, add 0.03 g of calcium carbonate (CaCO3), and sonicate for 30 minutes to ensure the complete dissolution of CaCO3.

[0054] Slowly add Solution B to Solution A and continue stirring for 2 hours to obtain a sol solution. Next, add 2 mL of polyvinyl butyral (PVB)-ethanol solution (6% w / v) to the above sol solution to obtain a mixed spinning solution for introducing phase separation for electrospun fibers. Stir the obtained mixed spinning solution for 36 hours and continuously evaporate the solvent to increase the viscosity of the sol. When the total liquid volume is reduced to 20 mL, transfer the aged mixed spinning solution into a plastic syringe equipped with a metal needle (21-gauge needle, 0.5 mm in diameter), apply a high-voltage electric field of 15 kV to the metal needle and the collecting plate for electrospinning. Then, transfer the bioactive glass membrane precursor obtained by electrospinning to an oven at 50°C and bake for 12 hours to evaporate the residual solvent, and then perform heat treatment, that is, heat to 700°C in an ammonia atmosphere and hold for 1 h to obtain the bionic periosteum containing CaMoO4 and Mo3N2.

[0055] Example 2

[0056] 1. Preparation of Bionic Periosteum

[0057] Preparation of Bionic Periosteum (CaMoO4 / Mo3N2-BGFM):

[0058] Based on the principle that partial hydrolysis of alkoxysilane produces linear aggregates, the bionic periosteum is prepared by the sol-gel method with the addition of phosphomolybdic acid and the 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 a 20 wt% ethanol solution of phosphomolybdic acid, mix well, then add 0.01 g of CaCO3, and sonicate for 30 minutes to ensure complete dissolution of CaCO3.

[0061] Slowly add Solution B to Solution A and continue stirring for 2 hours to obtain a sol solution. Next, add 2 mL of a polyvinyl butyral (PVB)-ethanol solution (6% w / v) to the above sol solution to obtain a mixed spinning solution for introducing phase separation for electrospun fibers. Stir the resulting mixed spinning solution for 36 hours to continuously evaporate the solvent to increase the viscosity of the sol. When the total liquid volume is reduced to 20 mL, load the aged mixed spinning solution into a plastic syringe equipped with a metal needle (21-gauge needle, 0.5 mm in diameter), apply a high-voltage electric field of 15 kV to the metal needle and the collection plate for electrospinning. Then, transfer the obtained bioactive glass membrane precursor by electrospinning to an oven at 25°C and bake for 12 hours to evaporate the residual solvent, and then perform heat treatment, that is, heat to 600°C in an ammonia atmosphere and hold for 0.8 h to obtain a biomimetic bone membrane containing CaMoO4 and Mo3N2.

[0062] Example 3

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

[0064] The present invention utilizes the principle of partial hydrolysis of alkoxysilane to generate linear aggregates, and prepares a biomimetic bone membrane through 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 a 20 wt% ethanol solution of phosphomolybdic acid, mix well, then add 0.05 g of CaCO3, and sonicate for 30 minutes to ensure complete dissolution of CaCO3.

[0067] Solution B was slowly added dropwise to Solution A, and stirring was continued for 2 hours to obtain a sol solution. Next, 2 mL of polyvinyl butyral (PVB)-ethanol solution (6% w / v) was added to the above sol solution to obtain a mixed spinning solution for introducing phase separation for electrospun fibers. The obtained mixed spinning solution was stirred for 36 hours, and the solvent was continuously volatilized 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 in diameter), and a high-voltage electric field of 15 kV was applied to the metal needle and the collecting plate for electrospinning. After that, the bioactive glass membrane precursor obtained by electrospinning was transferred to an oven at 100 °C (the temperature can be controlled at 25 - 100 °C) and baked for 12 hours to volatilize the residual solvent, and then heat treatment was carried out, that is, heated to 650 °C in an ammonia atmosphere and held for 0.5 h to obtain a bionic bone membrane containing CaMoO4 and Mo3N2.

[0068] Example 4

[0069] I. Experimental method

[0070] The following takes the calcium molybdate bionic bone membrane (CaMoO4-BGFM) and molybdenum nitride bionic bone membrane (Mo3N2-BGFM) as examples, and the bionic bone membrane prepared in Example 1 is used for characterization and performance description.

[0071] The preparation method of the calcium molybdate bionic bone membrane (CaMoO4-BGFM) is the same as that in Example 1, except that its heat treatment is carried out by heating to 800 °C in an air atmosphere and holding for 2 hours.

[0072] The preparation method of the molybdenum nitride bionic bone membrane (Mo3N2-BGFM) is the same as that in Example 1, except that its heat treatment is carried out by heating to 800 °C in an ammonia atmosphere and holding for 2 hours.

[0073] 1. Characterization of bionic bone membrane

[0074] The morphology and elemental composition of the antibacterial bionic bone membrane were studied using a field scanning electron microscope and energy-dispersive X-ray spectroscopy (EDS). The phase composition was measured by X-ray diffraction (XRD).

[0075] To detect the ability of different biomaterials (CaMoO4, Mo3N2, CaMoO4+Mo3N2, and CaMoO4 / Mo3N2) to react with peroxidase (POD) catalyzing H2O2, the method is as follows: Add 10 μL of the biomaterial dispersion (CaMoO4, Mo3N2, or CaMoO4 / Mo3N2, all at a concentration of 20 mg / mL) and 40 μL of H2O2 (100 mM) to 1.95 mL of acetic acid-sodium acetate buffer (0.1 M, pH = 5.0). For the CaMoO4+Mo3N2 group, add 10 μL each of the CaMoO4 dispersion (20 mg / mL) and the Mo3N2 dispersion (20 mg / mL) to the reaction system (by reducing the buffer to ensure a total reaction system of 2 mL). Subsequently, add 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB, 20 mM) to the above mixture. A color reaction is generated by POD, and the absorbance in the wavelength range of 400 - 800 nm is recorded using ultraviolet-visible absorbance to observe the generation amount of the TMB oxidation product (oxTMB). At the same time, set a blank control (CN), that is, replace the biomaterial dispersion with an equal amount of blank solvent.

[0076] 2. Biocompatibility of the antibacterial bionic periosteum

[0077] 2.1 Scanning electron microscope

[0078] Inoculate bone marrow mesenchymal stem cells (BMMSCs) on the surfaces of different bionic periosteums. After fixation, dehydration, and gold spraying, use a scanning electron microscope to observe the adhesion state of BMMSCs and the bionic periosteum.

[0079] 2.2 Cell viability and toxicity detection

[0080] Stain the cells using a cell viability / cytotoxicity staining kit (Beyotime, China). Inoculate BMMSCs on the surfaces of different antibacterial bionic periosteums and continuously culture for 48 hours. Add the Calcein AM / PI detection working solution to the cell culture dish and incubate at 37°C for 30 min, then observe the cell fluorescence under an inverted fluorescence microscope.

[0081] 3. Osteogenic tests in vitro and in vivo

[0082] 3.1 ROS level detection

[0083] After culturing BMMSCs on different biomaterials, collect the cells, stain them using a ROS detection kit (Beyotime, China), detect the ROS level of the cells using a flow cytometer (BD, USA), and analyze the results using FlowJo_V10.

[0084] 3.2 Immunofluorescence detection

[0085] After culturing BMMSCs on different biomaterials, the cells were fixed with 4% paraformaldehyde, washed 3 times with PBS, permeabilized with 0.5% Triton X-100 for 15 min, blocked with 20% goat serum, and then incubated with the primary antibody at 4°C overnight. After co-incubation with the fluorescent secondary antibody, the cell cytoskeleton and nucleus were stained with phalloidin and DAPI staining solution respectively, and the results were immediately observed under an inverted fluorescence microscope.

[0086] 3.3 Alkaline phosphatase (ALP) staining

[0087] After 7 days of continuous osteogenic induction, an alkaline phosphatase staining kit (Beyotime, China) was used to detect the ALP content. After fixing the cells, the staining working solution was added to the cell culture dish. After reacting for 1 hour, the reaction was terminated with deionized water, and the cells were 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 staining solution (Solarbio, China) for 1 hour. After washing away the excess dye with PBS, the cells were observed under an inverted microscope.

[0090] 3.5 Establishment of rat femoral defect model

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

[0092] 3.6 Micro-CT detection

[0093] At the 4th and 8th weeks after the successful establishment of the bone defect model, the rats were euthanized, the femurs were carefully removed and fixed in 4% paraformaldehyde for 24 hours, and the tissues were scanned using a high-resolution micro-CT scanner (Perkin Elmer, Japan). The parameters were adjusted to a voltage of 90 kV, 88 μA, and a resolution of 7 μm per pixel. The images were reconstructed using CTAn software (Perkin Elmer, Japan), and the parameters of the samples were analyzed using CTVox software (Perkin Elmer, Japan).

[0094] 3.7 Histological staining

[0095] At the 8th week after surgery, the rats were euthanized and the femurs were isolated. After fixation in 4% paraformaldehyde for 24 hours, they were treated with ethylenediaminetetraacetic acid (EDTA) decalcifying solution for 2 weeks. The samples were embedded in paraffin and cut into 5-μm sections. The sections were stained using an H&E staining kit (Solarbio, China) and a Masson staining kit (Solarbio, China), and observed using an inverted microscope.

[0096] 3.8 Immunohistochemical staining

[0097] For immunohistochemical staining analysis, the dewaxed sections were treated with 3% H2O2 for 5 minutes, and then with 5% BSA for 10 minutes. Then the sections were incubated with the primary antibody overnight at 4 °C. Subsequently, they were incubated with a biotin-conjugated secondary antibody, and visualized using the streptavidin-biotin staining technique. The cell nuclei were stained with hematoxylin, and the slides were photographed using a microscope.

[0098] 4 In vitro and in vivo antibacterial tests

[0099] 4.1 Establishment of a rat femoral defect infection model

[0100] After anesthetizing the rats with sodium pentobarbital, 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 femur surface was removed, and a single cortical bone defect with a diameter of 4 mm was made above the intercondylar of the femur using a sterile trephine. To establish the bone defect infection model, the present invention filled the bone defect site with a gelatin sponge immersed in a methicillin-resistant Staphylococcus aureus (MASR) suspension. In the experimental group, different biomimetic periosteums (CaMoO4, MO3N2, or CaMoO4 / MO3N2) were covered on the bone defect site, while the control group was not covered; each group was sutured layer by layer. Among them, the MO3N2+NIR and CaMoO4 / MO3N2+NIR groups were also set up, that is, photothermal reaction treatment was added on the basis of covering the biomimetic periosteum, and the laser power was 0.8 W / cm 2 , and the treatment time was 10 min.

[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. Then the body temperature was routinely monitored every day for 2 weeks after continuous treatment. Venous blood was obtained from the rats on the 3rd, 7th, and 14th days after treatment. Standard blood tests were performed to evaluate the systemic infection level of these rats.

[0103] 4.3 Tissue colony plate test

[0104] On the 3rd day of treating the infection model, the wound was opened again, and bone marrow was obtained from the femur, immediately placed in 1 mL of sterile physiological saline and shaken well. After dilution, it was spread evenly on LB agarose gel and incubated at 37 °C for 12 hours, and then the CFU value was calculated.

[0105] 4.4 H&E staining and Giemsa staining

[0106] On the 14th day of treating the infection model, the rats were euthanized, and the femurs were taken. After fixation with 4% paraformaldehyde and decalcification with EDTA, tissue sections were made. The tissue sections were stained using a Giemsa staining kit (Solarbio, China). Observation was performed using an inverted microscope.

[0107] 5. Statistical analysis

[0108] All data are presented as mean ± standard deviation (SD). All experiments were repeated at least 3 times. Student's t-test was used for comparison between two groups, and one-way analysis of variance (ANOVA) was used for comparison among multiple groups. Statistical significance was set at P < 0.05.

[0109] II. Experimental results

[0110] 1. Characterization results of the antibacterial bionic periosteum

[0111] As Figure 1 shown, all three bionic periosteums exhibited good continuity, with fiber diameters of approximately 800 - 1000 nm, and porous characteristics were shown inside the fibers. This microstructure is conducive to the degradation of the fibers. It can be speculated that the heterojunction nanozymes generated by degradation can be phagocytosed by bacteria, producing various catalytic effects to generate ROS, inhibiting the growth and reproduction of bacteria.

[0112] As Figure 2 shown, the elemental distribution of CaMoO4 / Mo3N2 - BGFM fibers was uniform, demonstrating the uniformity of the spinning solution sol. The resulting heterojunction nanozymes were uniformly distributed in the fibers, which is conducive to improving their catalytic activity.

[0113] As Figure 3 shown, the three bionic periosteums had the phases of CaMoO4, Mo3N2, and CaMoO4 / Mo3N2 respectively, proving the successful preparation of bioactive glass fibers with a CaMoO4 / Mo3N2 heterostructure.

[0114] 2. Detection results of POD enzyme activity

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

[0116] 3. Biocompatibility test results of the bionic periosteum

[0117] Under scanning electron microscopy, a continuous contact area was observed between the cells and the material surface, showing good adhesion. And it was observed that the cells extended pseudopodia to actively contact the material, indicating that the cells could carry out normal physiological activities on the material surface ( Figure 5 in a)); the results of cell live / dead staining 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 extremely small and sparsely distributed, indicating that the material had very low toxicity to the cells ( Figure 5 in b)).

[0118] 4. Test results of the in vitro osteogenic function of the bionic periosteum

[0119] 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 ( Figures 6 - 7 ); higher positive rates and calcium salt depositions were also observed by alkaline phosphatase staining and alizarin red S staining ( Figures 8 - 9 ); it was measured by flow cytometry that the antibacterial bionic periosteum could significantly relieve the ROS level in bone marrow mesenchymal stem cells ( Figure 10 ). These results indicate that the CaMoO4 / MO3N2 bionic periosteum can effectively reduce the cell ROS level and promote osteogenic differentiation.

[0120] 5. Test results of the in vitro antibacterial function of the antibacterial bionic 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 survival rate of 97.84% in the control group. And the survival rates of E. coli were 81.05%, 81.39%, 51.93%, 57.29%, and 41.41% respectively, also lower than the survival rate of 97.84% in the control group (Figure 11 In a); The results of scanning electron microscopy showed that the MASR in the control group presented a typical spherical shape, E. coli presented a typical rod shape, and the two bacteria had plump morphologies. After co-culture with the bionic periosteum, MASR and E. coli lost their typical morphological characteristics, the bacterial cells were deformed or shrunk, and depressions or even perforations appeared on the surface. The bacterial state in the CaMoO4 / MO3N2 group was worse than that in the CaMoO4 group and the MO3N2 group, and stronger antibacterial efficacy was obtained through the photothermal reaction (CaMoO4 / MO3N2 + NIR group) ( Figure 11 In b); The results of ROS staining showed that the ROS level of bacteria co-cultured with the antibacterial bionic periosteum was higher than that in the control group, and this effect was most significant in the CaMoO4 / MO3N2 + NIR group ( Figure 11 In c). These results indicate that the CaMoO4 / MO3N2 bionic periosteum has good antibacterial function in vitro.

[0122] 6. Test results of the osteogenic function of the antibacterial bionic periosteum in vivo

[0123] Micro CT scan results showed that at the 4th week of bone defect healing, the sunken bone cortex and proliferated bone mass could still be observed at the bone defect sites in each group, but the implantation of the antibacterial bionic periosteum significantly promoted the repair of bone defects. At the 8th week, obvious bone defects could still be observed in the control group (Control), and the repair degree of bone defects in the CaMoO4 group and the MO3N2 group was significantly better than that in the control group. The surface of the bone defect in the CaMoO4 / MO3N2 group was significantly flatter than that in the control group, and it had a 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 ); The results of H&E staining showed that at the 8th week, the cortical bone in the control group was sparse and disordered, the cortical bone in the CaMoO4 group and the MO3N2 group was significantly more and ordered than that in the control group, and the cortical bone thickness in the CaMoO4 / MO3N2 group was uniform and dense ( Figure 13 ); OPN and BMP2 are key regulatory factors for bone matrix formation, mineralization, and bone remodeling. The results of immunohistochemical staining showed that the most positive foci were observed in the CaMoO4 / MO3N2 group, indicating that the bone repair mediated by the osteogenic differentiation of BMMSCs at the bone defect site in the CaMoO4 / MO3N2 group was more active ( Figure 14 ). These results indicate that the CaMoO4 / MO3N2 bionic periosteum can promote the repair of bone defects in vivo.

[0124] 7. Test results of the antibacterial function of the antibacterial bionic periosteum in vivo

[0125] As Figure 15As shown, the present invention monitored the body temperature changes of rats within 2 weeks. The results showed that the body temperatures of rats before surgery were all within the normal range, reached the peak on the first day after surgery, and there was no difference among groups. Subsequently, the body temperature records showed that compared with the control group (Control), the body temperature recovery rate of the experimental group using the bionic periosteum was faster. On the 10th day after surgery, the body temperature of the rats in the CaMoO4 / MO3N2 group had recovered to the pre-surgical level, while the control group did not reach the recovery level of the CaMoO4 / MO3N2 group on the 9th day until the 14th day after surgery. Subsequently, the present invention also monitored the changes in the routine blood test of rat venous blood. The results showed that on the first day after surgery, the white blood cells (WBC) in each group exceeded the normal value, and the WBC index of the control group was the highest, reaching 2.08×10 10 / L. The increase was mainly in granulocytes. The results of the routine blood tests on the 7th, 14th, and 21st days after surgery showed that implanting the bionic periosteum could significantly relieve the abnormal blood routine caused by infection, and this effect was most obvious in the CaMoO4 / MO3N2 group ( Figure 16 ). Subsequently, the present invention evaluated the degree of femoral infection in rats by Wright-Giemsa staining and immunohistochemical staining. The results of Wright-Giemsa staining showed that the positive foci of Giemsa staining were significantly reduced after implanting the bionic periosteum, and the effect was most obvious in the CaMoO4 / MO3N2 group ( Figure 17 ). Subsequently, in order to further evaluate the activity of neutrophils in the bone marrow cavity, the present invention performed immunohistochemical staining of myeloperoxidase (MPO) on 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. While the number of MPO-positive cells in the rats implanted with the bionic periosteum decreased significantly and the distribution range shrank, indicating that the material effectively inhibited the infiltration of neutrophils and the inflammatory response ( Figure 18 ).

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

[0127] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A preparation method of an antibacterial bionic periosteum, characterized in that, It includes the following steps: Add solution B to solution A, obtain a sol solution through mixing reaction, then add a polyvinyl butyral-ethanol solution to obtain a mixed spinning solution. After stirring to volatilize the solvent, electrospinning is carried out to obtain a bioactive glass film precursor; Perform a drying treatment on the bioactive glass film precursor. After volatilizing the residual solvent, perform a heat treatment in a reducing atmosphere of ammonia gas to obtain the antibacterial bionic bone membrane; The preparation method of solution A includes: Dissolve tetraethoxysilane and triethyl phosphate in a solvent, and then add Ca(NO3)2·4H2O to obtain solution A; The preparation method of solution B includes: Add deionized water and calcium carbonate to a phosphomolybdic acid solution, and after fully dissolving, solution B is obtained.

2. The preparation method according to claim 1, characterized in that, The concentration of the polyvinyl butyral-ethanol solution is 6% w / v.

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

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

5. The preparation method according to claim 1, characterized in that, The temperature of the heat treatment is 600 - 700 °C, and the time is 0.5 - 1 h.

6. The preparation method according to claim 1, characterized in that, 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.

7. The preparation method according to claim 1, characterized in that, 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.

8. An antibacterial bionic bone membrane prepared by the preparation method according to any one of claims 1 - 7.

9. Use of the antibacterial bionic bone membrane according to claim 8 in the preparation of surgical supplies for treating infectious bone defects.

10. A surgical supply for treating infectious bone defects, characterized in that, The active ingredient includes the antibacterial bionic bone membrane according to claim 8.

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