Preparation method and application of multistage bionic periosteum capable of regulating and controlling osteogenesis microenvironment

Abstract

The invention belongs to the field of biological materials and tissue engineering, and provides a preparation method and application of a multistage bionic periosteum capable of regulating and controlling an osteogenesis microenvironment, oxidized xyloglucan is dissolved in water, BMP2 is added for mixing reaction, and an OXG-BMP2 solution is obtained; the preparation method comprises the following steps: adding an OXG-BMP2 solution into dichloromethane, dropwise adding an emulsifier, and stirring to obtain an emulsion containing OXG-BMP2 microparticles; adding L-polylactic acid and N, N-dimethylformamide into the emulsion containing the OXG-BMP2 microparticles, and obtaining a core-shell structure spun yarn by using an electrostatic spinning technology; the method comprises the following steps: mixing TA-Ce nano-enzyme into I-type rat tail collagen, uniformly mixing, dropwise adding into core-shell structure spinning, and incubating to complete self-assembly of the bionic periosteum, thereby obtaining the multi-stage bionic periosteum capable of regulating and controlling the osteogenesis microenvironment. The multi-stage bionic periosteum can regulate and control the osteogenesis microenvironment and sequentially promote osteogenesis, and has great application prospects in the aspects of bone injury repair and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomaterials and tissue engineering, and particularly relates to a preparation method of a multi-level biomimetic periosteum capable of regulating osteogenic microenvironment and application thereof. BACKGROUND

[0002] Due to the rapid development of society, bone defects caused by high-energy injury have become a major health problem affecting global human health. Therefore, the regeneration and repair of bone defects is one of the major challenges faced by clinical orthopedics. Traditional autologous bone transplantation has problems such as damage to the donor site and limited source, while allogeneic bone transplantation has the risk of immune rejection. Periosteum, as a key tissue wrapping the surface of bone, is rich in blood vessels, nerves and osteogenic cells, and plays a core role in the bone regeneration process by dynamically regulating the local microenvironment. Therefore, developing a biomimetic periosteum material to simulate the structure and function of natural periosteum and achieve precise regulation of the osteogenic microenvironment is of great significance for accelerating bone defect repair.

[0003] Bone defect repair is in great demand. Given the important role of periosteum in bone defect repair, finding a periosteum substitute material as a biomimetic periosteum for bone repair is a good solution. Current periosteum substitute materials mainly include synthetic polymer membranes (such as polylactic acid and polycaprolactone) and natural polymer membranes (such as collagen and silk fibroin). Although these materials have certain biocompatibility, they are difficult to achieve controlled release of growth factors and microenvironment regulation, and cannot dynamically respond to biological signals during bone repair. SUMMARY

[0004] To solve the above technical problems, the present application provides a preparation method of a multi-level biomimetic periosteum capable of regulating osteogenic microenvironment and application thereof. The present application provides a method for preparing a multi-level biomimetic periosteum relying on electrospinning technology and collagen self-assembly principle, thereby achieving the purpose of improving the osteogenic microenvironment and ultimately sequentially promoting bone repair. Furthermore, the present application also provides biological applications of the multi-level biomimetic periosteum.

[0005] A preparation method of a multi-level biomimetic periosteum capable of regulating osteogenic microenvironment, comprising the following steps: dissolving oxidized xyloglucan in water, adding BMP2 and mixing the reaction to obtain an OXG-BMP2 solution; adding the OXG-BMP2 solution to dichloromethane, adding an emulsifying agent dropwise, and stirring to obtain an emulsion containing OXG-BMP2 micro-particles; adding left-handed polylactic acid and N, N-dimethylformamide to the emulsion containing OXG-BMP2 micro-particles, and using electrospinning technology to obtain a core-shell structure spinning; mixing TA-Ce nanoscale enzyme into type I rat tail collagen, mixing uniformly, and adding dropwise to the core-shell structure spinning, and after incubation, completing self-assembly of the biomimetic periosteum to obtain a multi-level biomimetic periosteum capable of regulating osteogenic microenvironment.

[0006] Further, the preparation method of the TA-Ce nanoscale enzyme comprises the following steps: completely dissolving a surfactant in an ethanol aqueous solution, adding ammonia water and tannic acid and stirring overnight at room temperature, finally mixing with cerium acetate to perform hydrothermal reaction, and obtaining the TA-Ce nanoscale enzyme.

[0007] Further, the hydrothermal reaction condition is 100-120 DEG C for 6-10 hours.

[0008] Further, the preparation method of the oxidized xyloglucan comprises the following steps: dissolving xyloglucan in water, adding sodium periodate to perform oxidation, using ethylene glycol to terminate the reaction, and performing dialysis and freeze-drying to obtain the oxidized xyloglucan.

[0009] Further, the electrospinning parameter is set as follows: the flow is 0.4-0.6 mL / h, the voltage is 13-17 kV, the collector rotation speed is 80-120 rpm, and the distance between the needle tip and the collector is 10-15 cm.

[0010] Further, the dissolution mass-volume ratio of the TA-Ce nanoscale enzyme is 40-60 mu g / mL, and the ratio of the TA-Ce nanoscale enzyme to the core-shell structure spinning is 80-120 uL / cm. 2 .

[0011] Further, the incubation temperature is 35-40 DEG C, and the time is more than 30 min.

[0012] Further, the OXG-BMP2 solution is added into dichloromethane, an emulsifier is added dropwise, stirring is performed at room temperature for more than 20 min at 3000-4000 rpm to obtain an emulsion containing OXG-BMP2 micro-particles.

[0013] The application further provides a multi-level bionic bone membrane capable of regulating an osteogenic microenvironment, which is prepared by the preparation method.

[0014] The application further provides application of the multi-level bionic bone membrane capable of regulating an osteogenic microenvironment in improving an osteogenic microenvironment, promoting generation of an osteogenic related blood vessel and promoting a bone healing process.

[0015] One of the purposes of the application is to provide a preparation method of a multi-level bionic bone membrane capable of regulating an osteogenic microenvironment.

[0016] (1) completely dissolving a surfactant F127 in a 50% ethanol solution, adding ammonia water and tannic acid and stirring overnight at room temperature, finally adding cerium acetate into a hydrothermal reaction kettle, heating and reacting to obtain a novel nanoscale enzyme;

[0017] (2) dissolving xyloglucan (XG) in water in a mass-volume ratio of 1%, adding sodium periodate to perform oxidation, using ethylene glycol to terminate the reaction, and performing dialysis and freeze-drying to obtain oxidized xyloglucan (OXG).

[0018] (3) The oxidized xyloglucan (OXG) obtained in step (2) is dissolved in water at a mass / volume ratio of 1%, and BMP2 (10 μg / ml) is added to obtain a uniform OXG-BMP2 solution at a mass / volume ratio of 1%;

[0019] (4) The solution in step (3) is added to dichloromethane, and two drops of Span-80 are added dropwise, and high-speed stirring is performed for about 20 minutes to prepare an emulsion containing uniform OXG-BMP2 micro-particles;

[0020] (5) PLA and DMF are added to the emulsion in step (4) to obtain a core-shell structure spinning using an electrospinning technique;

[0021] (6) The nano-enzyme in step (1) is mixed into type I collagen, and the mixture is added dropwise to the core-shell spinning in step (5), and self-assembly of the biomimetic bone membrane is completed at 37°C for more than half an hour, that is, a multi-level biomimetic bone membrane is obtained.

[0022] The electrospinning technique is a special form of electrostatic atomization of a polymer fluid and a fiber manufacturing process. A polymer or melt can be jet-spun in a strong electric field to produce polymer filaments with a nanometer diameter. The electrospinning technique has the advantages of simple manufacturing device, low spinning cost, a wide variety of spinnable substances, controllable process, etc., and has become one of the main ways to effectively prepare nanofiber materials. The nanofiber prepared by the electrospinning technique has the advantages of good biocompatibility, large specific surface area, and high porosity. Therefore, it has a wide application in the fields of drug controlled release, wound repair, and biological tissue engineering in the biomedical field. The present application relies on the electrospinning technique, and uses a water-in-oil (W / O) emulsion of micro-sol particles to prepare a nanospinning membrane with a core-shell structure. The spinning membrane has better biocompatibility, better mechanical properties, and higher porosity, and can simultaneously load water-soluble drugs and fat-soluble drugs and achieve ordered release of the drugs, thereby achieving sequential treatment of diseases.

[0023] The collagen self-assembly principle refers to that collagen with a triple helix structure can also self-assemble into collagen fibers or collagen gels under suitable concentration, temperature, and pH conditions. The self-assembled products have a significant improvement in mechanical properties, thermal stability, and biological properties. The present application uses the collagen directional self-assembly principle, disperses the nano-enzyme in a type I collagen mixture which is the main component of the bone membrane extracellular matrix, and coats the type I collagen on the core-shell structure spinning membrane to locally regulate the osteogenic microenvironment. Then, the TA-Ce nano-enzyme is carried on the surface of the core-shell spinning material by coating the type I collagen (the main component of the bone membrane extracellular matrix) on the surface, so that the final multi-level biomimetic bone membrane material is obtained.

[0024] Natural extracts are widely used in biomaterials due to their excellent biocompatibility and antioxidant activity, and metal-phenolic networks (MPNs) have a wide range of applications in the construction of biomedical materials, which are composed of natural polyphenols and various metal ions, thereby providing functional adjustability by changing the coordinated metal ions, and integrating the advantages of nanozymes and non-enzymatic antioxidants. CeO2, as an excellent antioxidant nanozyme, has multiple catalytic properties, which can consume ROS and its downstream products by Ce 3+ and Ce 4+ redox state unique shuttle, regulate the microenvironment pH, thereby promoting the activation of osteoblasts and accelerating the bone repair process. The present application uses Ce ions to coordinate with natural polyphenol compounds tannic acid (TA) to synthesize a new antioxidant network nanozyme (TA-Ce). It has better biocompatibility, integrates the advantages of nanozymes and non-enzymatic antioxidants through metal-phenolic networks, realizes antioxidant cascade nanozymes with multiple ROS scavenging effects, and is used to improve the osteogenic microenvironment and promote bone repair.

[0025] Further, the dissolution sequence of the one standard sample in step (1) is 0.2 g F127 dissolved in 37 ml deionized water and 37 ml anhydrous ethanol, then 0.4 ml ammonia water and 0.2 g tannic acid are added, and the hydrothermal reaction condition is 110°C for 8 hours.

[0026] Further, the cerium acetate solution concentration in step (1) is 0.13 g cerium acetate dissolved in 2 ml deionized water, mixed with the previous tannic acid solution, stirred overnight at room temperature, and then added to the hydrothermal reactor.

[0027] Further, the stirring speed in step (4) is 3000-4000 rpm.

[0028] Further, the electrospinning parameter settings in step (5) are flow rate 0.5 ml / h, voltage 15 kV, collector speed 100 rpm, and distance between needle tip and collector 10-15 cm.

[0029] Further, the dissolution mass-volume ratio of the nanozyme in step (6) is 50 μg / ml, and the spinning drop amount relative to the nanozyme and type I mouse tail collagen mixed solution obtained in step (6) is 100 ul / cm 2 .

[0030] The second object of the present application is to provide a multi-level biomimetic bone membrane prepared by the above method, wherein the TA-Ce nanoscale enzyme has a needle shape, the content of cerium is 4-5wt%, the final biomimetic bone membrane has a spinning diameter of 2-3um, the outer collagen nanoscale enzyme layer improves the osteogenic microenvironment, the middle electrospun shell layer provides a cell scaffold, and the inner OXG-BMP2 loading promotes osteogenesis through the osteogenesis-vascular coupling effect.

[0031] The third object of the present application is to provide the application of the multi-level biomimetic bone membrane prepared by the above method in bone defect repair, which can improve the osteogenic microenvironment, promote the generation of osteogenesis-related blood vessels, and sequentially promote the bone healing process.

[0032] The present application is centered on the construction of an artificial biomimetic bone membrane for promoting bone defect repair, and a multi-level biomimetic bone membrane capable of regulating the osteogenic microenvironment is designed by using molecular biology and tissue engineering related technologies. The nanoscale enzyme removes early active oxygen, the type I collagen cooperates with the PLA electrospun shell to provide a cell scaffold, the core OXG-BMP2 controls the release of BMP2 and promotes late osteogenesis, and at the same time, promotes the generation of blood vessels through OXG. In an example of the present application, the rapid healing of bone defects can be successfully achieved.

[0033] Advantages

[0034] In a critical rat skull defect model, the present application product can improve the ratio of new bone volume / defect bone volume from 15% to about 50% compared with the ordinary collagen assembled PLA biomimetic bone membrane, thus proving that the present application product has great advantages in bone defect repair. Figure 9 .

[0035] The present application mixes cerium acetate, tannic acid, a surfactant and the like to obtain a uniform solution, heats the solution in a hydrothermal reaction kettle to obtain a new cascade nanoscale enzyme (TA-Ce NMs), uses polylactic acid (PLA) as an outer layer and oxidized xylan-bone morphogenetic protein 2 (OXG-BMP2) as an inner core to prepare a core-shell structure fiber (POB, PLA / OXG-BMP2) by electrospinning technology, and combines the nanoscale enzyme with the core-shell structure fiber by using the self-assembly characteristics of type I collagen (Collagen) to obtain a multi-level biomimetic bone membrane NMC@POB (TA-Ce NMs / Collagen / PLA / OXG-BMP2) capable of regulating the osteogenic microenvironment. The multi-level biomimetic bone membrane obtained by the method of the present application has the characteristics of simple preparation and good biocompatibility, and the diameter of the multi-level biomimetic bone membrane is 2-3um and is uniformly distributed.

[0036] The multi-stage biomimetic bone membrane obtained by the method has fiber silk with a three-layer structure, which is a collagen layer loaded with TA-Ce nanometer enzyme as an outer layer, electrospun PLLA as an intermediate layer, and OXG-BMP2 as an inner layer.

[0037] The multi-stage biomimetic bone membrane obtained by the method can improve the local osteogenic microenvironment and then release the inner layer drug to promote osteogenesis, and can load different types of drugs and bioactive factors in the later stage, so as to realize the sequential treatment effect of bone defects. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 It is a synthesis schematic diagram of oxidized xyloglucan;

[0039] Figure 2 It is a physical diagram of an electrospinning device;

[0040] Figure 3 It is the characterization result of TA-Ce nanometer enzyme; wherein A is scanning electron microscope element analysis; B is Fourier infrared spectrum (FT-IR) result; C-E are X-ray photoelectron spectrograms;

[0041] Figure 4 It is a transmission electron microscope image of simple PLA (P) spinning and OXG-BMP2 wrapped core-shell structure PLA spinning (POB);

[0042] Figure 5 It is the SEM characterization result of different stages of the biomimetic bone membrane;

[0043] Figure 6 It is the proliferation experiment result of the biomimetic bone membrane loaded with mesenchymal stem cells; A is the live and dead staining of cells planted on C@P, NMC@P, and NMC@POB three groups of biomimetic bone membranes, B is the skeleton staining and focal adhesion protein immunofluorescence staining of cells planted on the three groups of bone membranes;

[0044] Figure 7 It is the osteogenesis effect detection result of the biomimetic bone membrane; A is alkaline phosphatase staining, B is alizarin red staining;

[0045] Figure 8 It is the skeleton staining of the biomimetic bone membrane co-cultured with human umbilical vein endothelial cells;

[0046] Figure 9 It is the effect diagram of the biomimetic bone membrane on the bone repair of a rat skull defect; A is the three-dimensional reconstruction diagram of the new bone by Mmicro-CT in the bone defect area, B is the volume ratio quantitative analysis of the new bone volume / defect site bone volume (BV / TV). DETAILED DESCRIPTION

[0047] In order to make the purposes, technical solutions and advantages of the present application clearer and more apparent, the present application is described in detail below with reference to examples. It is necessary to point out that the following examples are only used to explain and illustrate the present application and do not limit the present application. Some non-essential improvements and adjustments made by those skilled in the art based on the above summary of the application still fall within the protection scope of the present application.

[0048] In the following examples, part of the experimental materials involved are as follows:

[0049] Polyether F127, tannic acid, cerium acetate trihydrate, Span 80, dichloromethane and N, N-dimethylformamide were purchased from Sigma-Aldrich Company in the United States, dextran was purchased from Van Der Company in China, sodium periodate was purchased from Aladdin Chemical Reagent Co., Ltd., PLLA polylactic acid was purchased from Jinan Daigang Biotechnology Co., Ltd. in China, and mouse tail type I collagen was purchased from Corning Company in the United States (item number 364236).

[0050] Example 1

[0051] Preparation and characterization of TA-Ce nanoscale enzyme

[0052] (I) Preparation of TA-Ce nanoscale enzyme

[0053] 0.2 g of F127 was weighed and dissolved in 37 ml of deionized water and 37 ml of anhydrous ethanol, 0.4 ml of ammonia water and 0.2 g of tannic acid were added and completely dissolved; 0.13 g of cerium acetate was weighed and dissolved in 2 ml of deionized water, and after complete dissolution, the two were mixed, stirred at room temperature overnight, and then added to a hydrothermal reaction kettle, which was maintained at 110°C for 8 hours. When washing the product, centrifugation at 14800 rpm for 5 minutes at room temperature was used to remove the supernatant, and the precipitate was resuspended with ethanol and then centrifuged under the same conditions to remove the supernatant three times. The precipitate was resuspended with deionized water and then centrifuged under the same conditions to remove the supernatant three times, and then dried to obtain the reaction product.

[0054] (II) Characterization of TA-Ce nanoscale enzyme

[0055] In order to verify the synthesis effect of TA-Ce nanoscale enzyme, scanning electron microscope element analysis (SEM-Mapping) was used to analyze the element composition of the obtained product (see Figure 3 ). First, SEM showed that TA-Ce nanoscale enzyme was needle-shaped, and SEM-Mapping results showed that cerium elements were uniformly distributed in the structure of nanoscale enzyme, and the element ratio was about 4%. Fourier infrared spectroscopy (FT-IR) results showed that after the reaction of TA and cerium acetate, the coordination of cerium ions and hydroxyl groups changed the number of free hydroxyl groups, and destroyed the hydrogen bond network between molecules, and the width and intensity of the peak at 3208 cm -1 changed; secondly, the peak at 1671 cm -1The shift absorption at 1557 cm⁻¹ indicates a coordination relationship between cerium ions and the C=O group in TA, thereby affecting the electronic distribution and vibrational frequency of the C=O bond. -1 The peak shift at certain points indicates a weaker interaction between cerium ions and aromatic rings, affecting their vibrational modes. These spectral modifications confirm the successful coordination of cerium ions with the hydroxyl and carboxyl groups in TA. X-ray photoelectron spectroscopy (XPS) further validates the surface properties of TA-Ce. The high-resolution C 1s spectrum shows three peaks at approximately 284.20, 285.67, and 288.09 eV, corresponding to C=C, CC, CO, and O=CO, respectively. The high-resolution O 1s spectrum shows peaks at 530.92 eV (Ce-OH) and 532.47 eV (Ce-OC), indicating successful formation of Ce-polyphenol coordination bonds. The high-resolution three-dimensional Ce spectrum shows six distinct peaks, with binding energies at 884.8 eV and 903.5 eV belonging to Ce. 3+ The remaining peaks correspond to Ce. 4+ The hybrid valence state of this cerium ion endows TA-Ce nanozymes with superior antioxidant capabilities.

[0056] Example 2

[0057] Preparation of core-shell spinning

[0058] This invention utilizes coaxial electrospinning technology to prepare core-shell structured fibers. A schematic diagram of the apparatus is shown below. Figure 2 The process involves using a PLLA sol emulsion containing OXG-BMP2. Under the influence of an electric field, the OXG-BMP2 droplets overcome surface tension and are stretched into a conical shape. They are then sprayed to form a jet, and finally, the inner and outer solutions are stretched together to form a core-shell spun structure where the core layer is wrapped by the shell layer.

[0059] The specific manufacturing method of the above-mentioned core-shell structure spinning is as follows:

[0060] (1) Synthesis of oxidized xylo-glucan (OXG)

[0061] Weigh 0.1g of xyloglucan and dissolve it in 9.9g of deionized water. Add 0.2g of sodium periodate and stir at room temperature for two hours. Pour the resulting liquid into a dialysis bag with a molecular weight cutoff of 3000-4500 and dialyze for three days. On the fourth day, collect the liquid from the dialysis bag, pre-freeze at -80℃ overnight, and freeze-dry to obtain oxidized xyloglucan (OXG) material (see...). Figure 1 ).

[0062] (2) Preparation of electrospinning solution for core-shell spinning

[0063] Take 0.1 g OXG dissolved in 9.9 g deionized water, after complete dissolution, add 0.1 g BMP2 (Bone Morphogenetic Protein 2, BMP2 polypeptide synthesized by Qiangyao Biotechnology Co., Ltd., sequence (N→C) is KIPKASSVPTELSAISTLYL) and stir well to form a uniform OXG-BMP2 sol, add two drops of Span-80 (Span 80, sorbitan monoleate), stir at room temperature for 20 minutes above 3000-4000 rpm, and prepare a uniform dispersion suspension. Subsequently, add 1 g of PLA and 1 g of N, N-dimethylformamide (DMF), continue to stir at room temperature at a low speed of 500-600 rpm for 15 minutes to obtain a uniform electrospinning solution.

[0064] (3) Electrospinning

[0065] Preparation using a conventional electrospinning device (see Figure 2 ), set to flow 0.5 ml / h, voltage 15 kV, collector speed 100 rpm, distance between needle tip and collector 10-15 cm. The obtained spinning is vacuum dried overnight, and the next day it is sterilized by ultraviolet irradiation for 20 minutes and ready for use. Transmission electron microscopy verifies that the obtained spinning has a clear core-shell structure compared with ordinary PLA spinning (see Figure 4 ).

[0066] Example 3

[0067] Preparation and characterization of multi-level biomimetic bone membrane NMC@POB (TA-Ce NMs / Collagen / PLA / OXG-BMP2)

[0068] The present application utilizes the self-assembly principle of type I collagen and the core-shell structure electrospinning technology to prepare a multi-level biomimetic bone membrane, which has a three-layer structure, which is a collagen layer carrying TA-Ce nanoscale enzyme as the outer layer, electrospun PLLA as the middle layer, and OXG-BMP2 as the inner layer.

[0069] (I) Preparation of multi-level biomimetic bone membrane

[0070] The core-shell structure spinning (POB, PLA / OXG-BMP2) and the ordinary electrospinning (P, PLA) are compared, 50 μg of TA-Ce nanoscale enzyme is mixed into 1 ml of type I mouse tail collagen (Collagen Type I of Corning Company, product number 364236), and 100 μl of the mixture of TA-Ce nanoscale enzyme and type I mouse tail collagen is added dropwise per 1 cm 2 of electrospinning, incubate at 37°C for more than half an hour to complete the self-assembly of the biomimetic bone membrane, and then wash with sterile PBS three times to obtain the multi-level biomimetic bone membrane.

[0071] (II) Characterization of multi-level biomimetic periosteum

[0072] The morphology of the multi-level biomimetic periosteum was evaluated using scanning electron microscopy (SEM) (see [link]). Figure 5 Based on the incorporation of TA-Ce nanozymes and the core-shell structure loaded with OXG-BMP2, the biomimetic periosteum was divided into three groups: ① collagen-coated PLA fiber membrane (C@P), ② collagen-coated PLA fiber membrane (TA-Ce NMs) (NMC@P), and ③ collagen-coated core-shell fiber membrane containing OXG-BMP2 microsol (NMC@POB). The results showed that electrospinning yielded uniformly and randomly arranged spun fibers. Through self-assembly, a spiderweb-like type I collagen network was formed, which not only tightly bound TA-Ce NMs to the fiber membrane but also established a hierarchical structure.

[0073] Example 4

[0074] Biocompatibility testing of multi-level biomimetic periosteum

[0075] To assess the biocompatibility of the biomimetic periosteum, we seeded bone marrow mesenchymal stem cells (BMSCs) onto the surface of the biomimetic periosteum material and performed tests such as live and dead cell staining and cytoskeleton staining.

[0076] (a) Live and dead staining

[0077] Remove the frozen Calcein AM / PI Double Staining Kit, thaw at room temperature, and vortex to mix all reagents. Prepare the Calcein AM / PI staining working solution: After thawing at room temperature, add 10 μL Calcein AM Solution (100 μM) and 10 μL LPI Solution (750 μM) to 1 ml Calcein AM Assay Buffer, along with the vortex-mixed Calcein AM Solution and PI Solution. Carefully aspirate the culture medium from the adherent cells, add an appropriate amount of PBS to each well to wash the cells, remove the PBS, repeat the washing once, and aspirate the PBS again. Add the staining working solution at a ratio of 100 μL per well in a 96-well plate or 200 μL per well in a 24-well plate, and incubate at 37°C for 10–30 min. After incubation, observe the staining effect under a fluorescence microscope (Calcein shows green fluorescence; PI shows red fluorescence). The staining results for both live and dead cells indicate that the cells in the biomimetic periosteum culture group have good viability and good biocompatibility.

[0078] (ii) Skeletal staining

[0079] The culture solution in the well plate was sucked off, washed with PBS for 3 times, and finally the PBS was discarded; 4% paraformaldehyde was added for cell fixation, washed with PBS for 3 times after 20 minutes of room temperature fixation; the cells were treated with immunofluorescence blocking solution for 1 hour, and then the blocking solution was discarded; the prepared working solution of phalloidin was added into the well plate, incubated at room temperature for 30 minutes in the dark, washed with PBS for 3 times; 200 μL of 4,6-diamino-2-benzoindole dilactic acid ester (DAPI) was added to label the cell nucleus, incubated at room temperature for 5 minutes in the dark; the staining solution was removed, and washed with PBS for 3 times. After the incubation, the staining effect was observed under a fluorescence microscope (phalloidin was red fluorescence; DAPI was blue fluorescence). All experimental groups showed good cell morphology, and the above results confirmed that the biomimetic periosteum not only had excellent biocompatibility, but also could provide an ideal platform for cell growth and proliferation.

[0080] Example 5

[0081] Detection of osteogenic effect of multi-stage biomimetic periosteum

[0082] To evaluate the osteogenic promoting effect of the biomimetic periosteum on bone marrow mesenchymal stem cells (BMSCs) in the reactive oxygen species (ROS) microenvironment, the alkaline phosphatase (ALP) activity of the early osteogenic marker was detected and alizarin red staining was performed after the cells were cultured in the osteogenic induction medium containing 200 μM hydrogen peroxide (H2O2) for 7 days or 14 days.

[0083] (1) Alkaline phosphatase staining

[0084] According to the cell density of 5 × 10 4Cells were seeded on four types of nanofiber membranes: grouped as C@P, NMC@P, C@POB and NMC@POB, C@P as control. After two days of culture in a-MEM complete medium, the medium was replaced with osteogenic induction medium containing 200 μΜ hydrogen peroxide (H2O2), and when cultured to the 7th day, ALP staining and ALP quantification were performed. ALP staining: discard the culture medium, wash once with PBS. 4% PFA 200 μL / well, fix for 30 min. Discard the PFA, wash 3 times with PBS. ALP staining kit, prepare the staining reagent, 200 μL / well, avoid light incubation for 15 min. Discard the staining reagent, wash 3 times with PBS, observe under a microscope and take pictures. ALP quantification: lyse the cells with Western and IP cell lysis solution without EDTA, and extract the total intracellular protein. Prepare the standard working solution according to the instructions of the alkaline phosphatase, and then determine according to the instructions, incubate at 37°C for 10 min. Stop the reaction, measure the absorbance at 405 nm, and calculate the relative activity of ALP. After 7 days of osteogenic induction, ALP staining showed that the osteogenic effect of C@POB and NMC@POB was significantly enhanced, among which NMC@POB was the most obvious, confirming that without the use of TA-Ce nanenzyme or the addition of OXG-BMP2 component, the osteogenic effect was significantly insufficient.

[0085] (ii) Alizarin red staining

[0086] When the cells were cultured for 14 days, alizarin red staining was performed, and the steps were as follows: discard the culture medium, gently wash once with PBS. 4% PFA 200 μL / well, fix for 30 min. Discard the PFA, wash 3 times with deionized water. Add 200 μL of alizarin red staining solution to each well, and stain for 10 min. Discard the staining solution, wash 3 times with deionized water, and observe under a microscope and take pictures. After taking pictures, add 400 μL of 10% perchloric acid to each well, dissolve the calcium nodules at room temperature for 5 min, then transfer to a 96-well plate, 100 μL per well, and measure the absorbance at 420 nm wavelength with a microplate reader. After 14 days of induction, alizarin red S (ARS) staining showed that the C@P group and NMC@P only had a small amount of calcium nodules deposited, while the C@POB and NMC@POB groups had a significant increase in mineralization deposition, among which NMC@POB was the most obvious, confirming that without the use of TA-Ce nanenzyme or the addition of OXG-BMP2 component, the osteogenic effect was significantly insufficient.

[0087] Example 6

[0088] Detection of the angiogenic effect of the multi-stage biomimetic bone membrane

[0089] To verify the angiogenic effect of the multi-stage biomimetic periosteum, a capillary tube formation experiment was performed. Matrigel matrix glue (Corning, USA) and a 24-well plate were pre-cooled at 4°C. Human umbilical vein endothelial cells (HUVECs) were cultured overnight in high-glucose DMEM medium without fetal bovine serum (FBS). After thawing the Matrigel matrix glue, 20 μL was added to each pre-cooled 24-well plate to ensure that no air bubbles were formed. Then the culture plate was placed at 37°C for 30 minutes to solidify the matrix glue. According to the co-culture method, 5 × 10 3

[0001] Example 7

[0091] NMC@POB promotes rat skull defect effect detection

[0092] To verify the in vivo osteogenic effect of the Ce-TA nanose and OXG-BMP2 components in the product composition, a rat critical skull defect model was used for verification. The results show that the addition of Ce-TA nanose can promote bone repair in the early stage (4 weeks), while OXG-BMP2 significantly promotes the bone repair process in the later stage (8 weeks). Micro-CT quantitative analysis shows that the ratio of new bone volume to defect bone volume is increased from 15% to about 50% compared with ordinary collagen assembled PLA biomimetic periosteum, thus proving that the product has great advantages in bone defect repair (. Figure 9 ).​

Claims

1. A method for preparing a multi-stage biomimetic periosteum that can regulate the osteogenic microenvironment, characterized in that, The method comprises the following steps: The OXG-BMP2 solution is added into dichloromethane, an emulsifier is added dropwise, and after stirring, an emulsion containing OXG-BMP2 micro-particles is obtained; the left-handed polylactic acid and N, N-dimethylformamide are added into the emulsion containing OXG-BMP2 micro-particles, and a core-shell structure spinning is obtained by using an electrospinning technology; the TA-Ce nanometer enzyme is mixed into the type I mouse tail collagen, and after being mixed uniformly, the TA-Ce nanometer enzyme is added dropwise into the core-shell structure spinning, and after incubation, the self-assembly of the biomimetic bone membrane is completed, and a multi-level biomimetic bone membrane capable of regulating the osteogenic microenvironment is obtained.

2. The method of claim 1, wherein the preparation of the multi-stage biomimetic periosteum with controllable osteogenic microenvironment is characterized by, The preparation method of the TA-Ce nanometer enzyme comprises the following steps: after a surfactant is completely dissolved in an ethanol aqueous solution, ammonia water and tannic acid are added, and stirring is performed at room temperature overnight; finally, the TA-Ce nanometer enzyme is obtained by mixing and performing a hydrothermal reaction with cerous acetate.

3. The method for preparing a multi-level biomimetic periosteum with a controllable osteogenic microenvironment according to claim 2, characterized in that, The hydrothermal reaction condition is 100-120 DEG C for 6-10 hours.

4. The method of claim 1, wherein the preparation of the multi-stage biomimetic periosteum with controllable osteogenic microenvironment is characterized by, The preparation method of the oxidized xyloglucan comprises the following steps: xyloglucan is dissolved in water, sodium periodate is added for oxidation, ethylene glycol is used to terminate the reaction, and then the oxidized xyloglucan is obtained by dialysis and freeze-drying.

5. The method of claim 1, wherein the preparation of the multi-stage biomimetic periosteum with controllable osteogenic microenvironment is characterized by, The electrospinning parameter is set as follows: a flow rate of 0.4-0.6 mL / h, a voltage of 13-17 kV, a collector rotating speed of 80-120 rpm, and a distance between a needle tip and the collector of 10-15 cm.

6. The method of claim 1, wherein the preparation of the multi-stage biomimetic periosteum with controllable osteogenic microenvironment is characterized by, The TA-Ce nanosome has a ratio of solubility mass volume of 40-60 μg / mL, and the ratio of TA-Ce nanosome to core-shell structure spinning is 80-120 uL / cm 2 .

7. The method of claim 1, wherein the method is further defined by: The incubation temperature is 35-40 DEG C, and the time is more than 30 min.

8. The method of claim 1, wherein the preparation of the multi-stage biomimetic periosteum with controllable osteogenic microenvironment is characterized by, The OXG-BMP2 solution is added into dichloromethane, an emulsifier is added dropwise, and after stirring at room temperature for more than 20 min at a speed of 3000-4000 rpm, an emulsion containing OXG-BMP2 micro-particles is obtained.

9. A multi-stage biomimetic periosteum that can regulate the osteogenic microenvironment, characterized in that, The multi-level biomimetic bone membrane capable of regulating the osteogenic microenvironment is prepared by the preparation method in any one of claims 1-8. 10.The multi-level biomimetic bone membrane capable of regulating the osteogenic microenvironment of claim 9 is used for improving the osteogenic microenvironment, promoting the generation of osteogenic related blood vessels, and promoting the bone healing process.

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