A polydopamine and magnesium ion modified silk scaffold material and its preparation method and application

Abstract

The present invention provides a silk scaffold material modified with polydopamine and magnesium ions, and its preparation method and application, belonging to the technical field of bone repair materials. The present invention uses hot-pressed flat silk as the base material, eliminating a series of tedious steps such as cocoon peeling and degumming in traditional technologies. The operation is simple, the cost is low, and the process is environmentally friendly. After the flat silk is hot-pressed, the mechanical properties of the silk scaffold material are greatly improved. The present invention uses a ternary solution to micro-treat the silk fiber substrate, thereby increasing the surface roughness of the base material, which is conducive to the subsequent adhesion and growth of cells. Polydopamine formed by dopamine self-polymerization is then grafted onto the material surface, thereby improving the hydrophilicity and biocompatibility of the silk fiber substrate. Magnesium ions can replace the hydroxyl groups in the catechol structure of polydopamine. By utilizing the grafting effect of polydopamine, magnesium ions are grafted onto the surface of the silk fiber substrate, thereby exerting excellent effects in regulating macrophage polarization, promoting angiogenesis, and promoting bone regeneration.

Description

Technical Field

[0001] The present invention relates to the technical field of bone repair materials, and in particular to a polydopamine and magnesium ion modified silk scaffold material, a preparation method thereof, and an application thereof. Background Art

[0002] The incidence of bone injuries due to accidents and illness is increasing worldwide. Furthermore, with the continued growth of the global population, the proportion of the elderly population continues to increase, leading to a significant increase in the incidence of orthopedic diseases, and a consequent increase in the demand for and difficulty in bone repair. Currently, biomaterials used for bone repair are categorized into the following categories: medical bioceramics, medical polymer materials, medical composite materials, and nano-artificial bone.

[0003] Medical bioceramic materials mainly use hydroxyapatite (HAP) to repair bone tissue. Because its composition properties are very similar to HAP of biological hard tissue, it also has good biocompatibility and can form strong bone bonds with natural bone, which is beneficial for subsequent mineralization and bone tissue regeneration. However, its strength is relatively low and it is not suitable for use as a load-bearing component in the human body.

[0004] Polymers have been widely used as bone repair materials, among which degradable polylactic acid (PLA) is used in oral surgery, polymethyl methacrylate (PMMA) bone cement is used for bone filling, and polyglycolic acid (PGA) is used as absorbable screws for bone fixation. The elastic modulus of bone grafting materials made of biodegradable materials is closer to that of bone tissue than that of metals. As the fracture heals, the material gradually degrades in the body and does not require secondary surgery to remove it.

[0005] Silk, a natural polymer biomaterial, has established a strong reputation in bone tissue engineering applications due to its many unique properties, including excellent biocompatibility, biodegradability, mechanical behavior, and ease of processing. Through different processing techniques, silk fibroin can be processed into various biomaterials, including porous silk sponges, silk hydrogels, silk-based scaffolds, and silk nanoparticles. However, traditional applications of silk fibroin require a series of processing steps, which are complex, costly, and environmentally friendly, making widespread application difficult. Summary of the Invention

[0006] In light of this, the present invention aims to provide a polydopamine and magnesium ion-modified silk scaffold material, its preparation method, and its application. The preparation method provided by the present invention is simple to operate, low-cost, and environmentally friendly. The resulting silk scaffold material exhibits excellent mechanical properties and biocompatibility, and can regulate macrophage polarization, promote angiogenesis, and promote bone regeneration.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing a silk scaffold material modified with polydopamine and magnesium ions, comprising the following steps:

[0009] (1) hot pressing the stacked multi-layer flat silk to obtain a hot pressed silk fiber substrate;

[0010] (2) soaking the hot-pressed silk fiber substrate in a ternary solution, taking it out and freeze-drying it to obtain a pretreated silk fiber substrate; the ternary solution includes CaCl2, ethanol and water;

[0011] (3) Immersing the pretreated silk fiber substrate in a polydopamine solution containing magnesium ions for modification to obtain a silk scaffold material modified with polydopamine and magnesium ions.

[0012] Preferably, the stacking thickness of the multi-layer flat wire is 0.1 to 30 cm;

[0013] The hot pressing temperature is 80-130° C., the hot pressing pressure is 10-500 MPa, and the heat preservation and pressure holding time is 5-180 minutes.

[0014] Preferably, in the ternary solution, the molar ratio of CaCl2, ethanol and water is 0.9-1:2:6.5-8.

[0015] Preferably, the soaking temperature in step (2) is 60-100° C. and the soaking time is 5-15 minutes.

[0016] Preferably, the method for preparing the polydopamine solution containing magnesium ions comprises the following steps:

[0017] Dopamine hydrochloride is mixed with a Tris-HCl solution to undergo a self-polymerization reaction to obtain a polydopamine solution;

[0018] A soluble magnesium salt is mixed with the polydopamine solution to obtain a polydopamine solution containing magnesium ions.

[0019] Preferably, the concentration of the Tris-HCl solution is 10 mM and the pH value is 8.5.

[0020] Preferably, in the polydopamine solution containing magnesium ions, the concentration of magnesium ions is 0.00005 to 0.01 mol / L, and the concentration of polydopamine is 2 to 4 mg / mL.

[0021] Preferably, the modification time in step (3) is 12 to 24 hours.

[0022] The present invention provides a polydopamine and magnesium ion modified silk scaffold material prepared by the above preparation method, comprising a hot-pressed silk fiber substrate and polydopamine and magnesium ions modified on the surface of the silk fiber substrate.

[0023] The present invention provides the use of the polydopamine and magnesium ion modified silk scaffold material in the preparation of bone repair materials.

[0024] The present invention provides a method for preparing a silk scaffold material modified with polydopamine and magnesium ions, comprising the following steps: (1) hot-pressing stacked multi-layer flat silk to obtain a hot-pressed silk fiber substrate; (2) immersing the hot-pressed silk fiber substrate in a ternary solution, taking it out and freeze-drying it to obtain a pretreated silk fiber substrate; the ternary solution comprises CaCl2, ethanol and water; and (3) immersing the pretreated silk fiber substrate in a polydopamine solution containing magnesium ions to modify it and obtain a silk scaffold material modified with polydopamine and magnesium ions. The present invention uses hot-pressed flat silk as the base material, eliminating a series of tedious steps such as cocoon peeling and degumming in traditional technologies. It is simple to operate, low-cost, and environmentally friendly. After the hot-pressed flat silk, the mechanical properties of the silk scaffold material are greatly improved; the present invention uses a ternary solution to micro-process the silk fiber substrate, so that the surface roughness of the base material is increased, which is beneficial to the subsequent adhesion and growth of cells. Then, polydopamine formed by dopamine self-polymerization is grafted on the material surface by relying on its own adhesive protein-like structure, thereby improving the hydrophilicity of the silk fiber substrate and making it have better biocompatibility; magnesium ions can replace the hydroxyl groups in the catechol structure of polydopamine. The present invention utilizes the grafting effect of polydopamine to graft magnesium ions to the surface of the silk fiber substrate, thereby exerting excellent effects in regulating macrophage polarization, promoting angiogenesis and promoting bone regeneration. Mc3t3-e1 cell and macrophage proliferation experiments showed that the biocompatibility of the silk fiber substrate was improved after cross-linking with polydopamine; polarization experiments showed that the silk scaffold material modified with polydopamine and magnesium ions of the present invention can promote the phenotypic transformation of macrophages; Alizarin red and alkaline phosphatase staining experiments showed that the silk scaffold material modified with polydopamine and magnesium ions of the present invention has excellent osteogenesis ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a physical picture of the hot-pressed silk fiber substrate with different treatments;

[0026] Figure 2 The infrared spectra of hot-pressed silk fiber substrates with different treatments;

[0027] Figure 3 is the contact angle of hot-pressed silk fiber substrates with different treatments;

[0028] Figure 4The stress-strain change diagram of hot-pressed silk fiber substrates with different treatments;

[0029] Figure 5 The EDS element analysis diagram of hot-pressed silk fiber substrates with different treatments;

[0030] Figure 6 Quantitative diagram of magnesium element in hot-pressed silk fiber substrates with different treatments;

[0031] Figure 7 The results are for Mc3t3-e1 cell proliferation;

[0032] Figure 8 Raw cell proliferation results;

[0033] Figure 9 It is the result of macrophage polarization;

[0034] Figure 10 The results of Alizarin Red and Alkaline Phosphatase staining experiments;

[0035] Figure 11 This is the result of Micro-CT three-dimensional reconstruction image;

[0036] Figure 12 The figures are the quantitative results of bone volume fraction in mice 6 weeks after surgery. DETAILED DESCRIPTION

[0037] The present invention provides a method for preparing a silk scaffold material modified with polydopamine and magnesium ions, comprising the following steps:

[0038] (1) hot pressing the stacked multi-layer flat silk to obtain a hot pressed silk fiber substrate;

[0039] (2) soaking the hot-pressed silk fiber substrate in a ternary solution, taking it out and freeze-drying it to obtain a pretreated silk fiber substrate; the ternary solution includes CaCl2, ethanol and water;

[0040] (3) Immersing the pretreated silk fiber substrate in a polydopamine solution containing magnesium ions for modification to obtain a silk scaffold material modified with polydopamine and magnesium ions.

[0041] The present invention heat-presses stacked layers of flat silk to produce a hot-pressed silk fiber substrate. In the present invention, the flat silk is preferably produced by five-instar mature silkworms spinning on a two-dimensional spinning bed. In the present invention, the flat silk preferably has a gram weight of 2 to 3 grams.

[0042] In the present invention, the number of stacked layers of the flat yarn is preferably 1 to 300, more preferably 10 to 200, and even more preferably 50 to 100. In the present invention, the stacked thickness of the multi-layer flat yarn is preferably 0.1 to 30 cm, more preferably 1 to 15 cm, and even more preferably 5 to 10 cm.

[0043] The present invention preferably uses a hot press for the hot pressing. In the present invention, the hot pressing temperature is preferably 80-130°C, more preferably 100-120°C; the hot pressing pressure is preferably 10-500 MPa, more preferably 50-300 MPa, and even more preferably 100-200 MPa; the heat holding time is preferably 5-180 minutes, more preferably 10-150 minutes, and even more preferably 30-100 minutes. The present invention uses the hot pressing to thermoplastically shape the silk fiber substrate.

[0044] After obtaining the hot-pressed silk fiber substrate, the present invention soaks the hot-pressed silk fiber substrate in a ternary solution, removes the substrate, and freeze-dries the substrate to obtain a pretreated silk fiber substrate; the ternary solution includes CaCl2, ethanol, and water. In the present invention, the molar ratio of CaCl2, ethanol, and water in the ternary solution is preferably 0.9 to 1:2:6.5 to 8, more preferably 1:2:8, 1:2:7, 1:2:6.5, or 0.9:2:8.

[0045] In the present invention, the immersion temperature is preferably 60-100°C, more preferably 70-80°C, and the immersion time is preferably 5-15 minutes, more preferably 10 minutes. The present invention utilizes an appropriate ternary solution micro-processing time on the material surface to dissolve some of the sericin on the material surface in the ternary solution, resulting in an irregular surface texture. This, in turn, increases the roughness of the hot-pressed silk fiber substrate surface to a certain extent.

[0046] In the present invention, the freeze-drying preferably includes sequential freezing and vacuum freeze-drying. In the present invention, the freezing is preferably performed in a refrigerator, the freezing temperature is preferably -40°C to -100°C, and the freezing time is preferably 12 hours. The present invention has no special requirements for the specific operation method of the vacuum freeze-drying, and vacuum freeze-drying methods familiar to those skilled in the art can be used.

[0047] After obtaining the pretreated silk fiber substrate, the present invention immerses the pretreated silk fiber substrate in a polydopamine solution containing magnesium ions to modify it, thereby obtaining a silk scaffold material modified with polydopamine and magnesium ions. In the present invention, the method for preparing the polydopamine solution containing magnesium ions preferably includes the following steps:

[0048] Dopamine hydrochloride is mixed with a Tris-HCl solution to undergo a self-polymerization reaction to obtain a polydopamine solution;

[0049] A soluble magnesium salt is mixed with the polydopamine solution to obtain a polydopamine solution containing magnesium ions.

[0050] In the present invention, the concentration of the Tris-HCl solution is preferably 10 mM, and the pH value is preferably 8.5. In the present invention, the temperature of the self-polymerization reaction is preferably room temperature, and the time is preferably 12 to 24 hours, more preferably 16 to 20 hours.

[0051] In the present invention, the concentration of polydopamine in the polydopamine solution is preferably 2 to 4 mg / mL, more preferably 3 mg / mL.

[0052] In the present invention, the soluble magnesium salt is preferably MgSO4, and the concentration of magnesium ions in the polydopamine solution containing magnesium ions is preferably 0.00005 to 0.01 mol / L, more preferably 0.0001 to 0.0004 mol / L.

[0053] In the present invention, the order of soaking the pretreated silk fiber substrate in the polydopamine solution containing magnesium ions is preferably: first soaking the pretreated silk fiber substrate in the polydopamine solution, and then adding the soluble magnesium salt.

[0054] In the present invention, the modification temperature is preferably room temperature, and the modification time is preferably 12 to 24 hours, more preferably 16 to 20 hours. In the modification process, polydopamine is grafted onto the surface of the pretreated silk fiber substrate, and magnesium ions replace the hydroxyl groups in the catechol structure of dopamine, thereby achieving magnesium ion modification on the surface of the silk fiber substrate.

[0055] After the modification, the polydopamine and magnesium ion modified silk scaffold material is preferably washed and dried. In the present invention, the washing is preferably water washing, and the drying is preferably natural air drying.

[0056] The present invention provides a polydopamine and magnesium ion modified silk scaffold material prepared by the above preparation method, comprising a hot-pressed silk fiber substrate and polydopamine and magnesium ions modified on the surface of the silk fiber substrate.

[0057] The present invention provides the use of the aforementioned polydopamine and magnesium ion-modified silk scaffold material in the preparation of a bone repair material. The polydopamine and magnesium ion-modified silk scaffold material provided by the present invention exhibits excellent mechanical properties and biocompatibility, can regulate macrophage polarization, promote angiogenesis, and promote bone regeneration, and can be used as a repair scaffold material for bone defects in various locations.

[0058] The polydopamine and magnesium ion modified silk scaffold material provided by the present invention, its preparation method and application are described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0059] Example 1

[0060] A certain number of fifth-instar mature silkworms are placed on a two-dimensional spinning bed to spin silk, obtaining 0.1-0.3 cm flat silk. The obtained flat silk is stacked into multi-layer flat silk with a thickness of 2-4 layers, and the multi-layer flat silk is thermoformed using a hot press, wherein the hot pressing temperature is 105°C, the time is 1 hour, and the pressure is 100 MPa to form a hot-pressed silk fiber substrate.

[0061] The hot-pressed silk fiber substrate was pretreated in a ternary solution containing a molar ratio of CaCl2:EtOH:H2O of 1:2:8 at 80°C for 10 minutes. The pretreated hot-pressed silk fiber substrate was frozen in a refrigerator at -80°C for 12 hours and then vacuum freeze-dried to form a surface-treated hot-pressed silk fiber substrate.

[0062] Use 10mL Tris-HCl buffer (10mM, pH=8.5) as a solvent to dissolve 20mg of dopamine hydrochloride to obtain a polydopamine solution; the surface-treated hot-pressed silk fiber substrate is immersed in the polydopamine solution, and MgSO4 is added to control the concentration of MgSO4 in the polydopamine solution to 0.00005mol / L. After 24h, the material is taken out and washed three times with deionized water to form a silk scaffold material modified with polydopamine and magnesium ions, which is recorded as TH-PDA@Mg1.

[0063] Example 2

[0064] The difference from Example 1 is that the concentration of MgSO4 in the polydopamine solution is 0.0001 mol / L, which is recorded as TH-PDA@Mg2.

[0065] Example 3

[0066] The difference from Example 1 is that the concentration of MgSO4 in the polydopamine solution is 0.0002 mol / L, which is recorded as TH-PDA@Mg3.

[0067] Example 4

[0068] The difference from Example 1 is that the concentration of MgSO4 in the polydopamine solution is 0.0004 mol / L, which is recorded as TH-PDA@Mg4.

[0069] Example 5

[0070] The difference from Example 1 is that the concentration of MgSO4 in the polydopamine solution is 0.001 mol / L, which is recorded as TH-PDA@Mg5.

[0071] Test Example 1

[0072] In the following structural characterization and performance tests, HFSC represents hot-pressed silk fiber substrate; THFSC represents surface-treated hot-pressed silk fiber base; TH-PDA represents surface-treated hot-pressed silk fiber base after immersion in polydopamine solution; TH-PDA@Mg1-5 represents surface-treated hot-pressed silk fiber base after immersion in polydopamine solution at different magnesium ion concentrations.

[0073] Actual pictures of hot-pressed silk fiber substrates with different treatments Figure 1 As shown, the infrared spectrum is as Figure 2 From the actual image and infrared spectrum, it can be observed that dopamine successfully self-polymerizes into polydopamine and cross-links on the surface of the silk fiber substrate.

[0074] The contact angles of hot-pressed silk fiber substrates with different treatments are as follows: Figure 3 As shown in the stress-strain diagram, Figure 4 The contact angle and stress-strain data show that the polydopamine and magnesium ion modified silk fiber substrate has better hydrophilicity, which is conducive to the adhesion and growth of cells. At the same time, the mechanical properties of the hot-pressed silk fiber after surface treatment are also improved to a certain extent.

[0075] EDS test was carried out on the hot-pressed silk fiber substrates with different treatments, and the obtained EDS element analysis diagram is shown as follows: Figure 5 As shown, Figure 5 Red represents carbon, green represents nitrogen, blue represents oxygen, and yellow represents magnesium. The quantitative graph of magnesium in hot-pressed silk fiber substrates with different treatments is shown in Figure 2. Figure 6 shown.

[0076] Depend on Figure 5 and Figure 6 It can be seen that polydopamine is successfully used to graft magnesium ions on the surface of the silk fiber substrate and as the concentration of magnesium ions increases, the magnesium ions on the surface of the silk fiber substrate also increase.

[0077] Test Example 2 Mc3t3-e1 cell and macrophage proliferation experiment

[0078] The Mc3t3-e1 cells used in this test example were purchased from the Cell Bank of the Committee of Typical Culture Collection of the Chinese Academy of Sciences / Cell Resource Center of the Shanghai Institutes for Biological Studies of the Chinese Academy of Sciences; RAW264.7 cells were derived from this research group. Mc3t3-e1 cells were cultured in α-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2, with the medium replaced every 3 days; RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2, with the medium replaced every 3 days. Each group of silk fiber substrates was soaked in 75% alcohol for 2 to 3 hours, rinsed with PBS 3 to 5 times, and then placed in an ultraviolet environment for 2 to 3 hours. Mc3t3-e1 cells and RAW264.7 were cultured at a rate of 5×10 4 Cells were seeded at a density of 100 cells / well in a 48-well plate. After 24 hours, the above sterilized materials were placed in the well plate and cultured with the cells in an incubator. The MTS method was used to detect the proliferation activity of both cells. At the predetermined time point (24h48h), the cell plate was taken out, the culture medium was aspirated, and it was slowly rinsed with PBS. After adding 500μL of MTS working solution to each well, it was placed in a cell culture incubator and incubated in the dark for 2 hours. Finally, 100μL of supernatant was aspirated into a 96-well plate, and the absorbance at a wavelength of 490nm was detected by an enzyme reader. The culture medium containing 10% FBS was inoculated as a control, and its absorbance value was used to determine the 100% survival rate of the cells. Three biological replicates were set up for all experimental groups. The results are expressed as cell viability: Cell viability (%) = (A e -A n ) / (A p -A n )×100, where A p is the absorbance of the positive control group, A n is the absorbance of the negative control group, A e is the absorbance of the experimental group.

[0079] The results of Mc3t3-e1 cell proliferation were as follows Figure 7 The results of Raw cell proliferation are shown in Figure 8 The results showed that the biocompatibility of the silk fiber matrix was improved after cross-linking with polydopamine.

[0080] Test Example 3 Macrophage Polarization Experiment

[0081] Macrophages, as a precursor cell type that triggers the innate immune response, possess unique functional and phenotypic characteristics. Based on their function and surface markers, macrophages can be divided into the classically activated M1 phenotype and the alternatively activated M2 phenotype. M1 macrophages are generally considered to express high levels of proinflammatory cytokines, such as nitric oxide synthase (iNOS), vascular endothelial growth factor (VEGF), interleukin IL-6, and tumor necrosis factor TNF-α, inducing inflammatory infiltration. M2 macrophages typically possess anti-inflammatory molecule signatures, such as the mannose receptor MRC-1 (also known as CD206), chemokines, and transglutaminase 2. Macrophages aggregate at the implant site and adhere to the implanted material to form giant cells. They secrete various chemical components to regulate the microenvironment and play a crucial role in tissue regeneration. Studies have reported that biomaterial-induced M2 macrophage polarization facilitates rapid repair of bone injury regeneration.

[0082] In this test, the effect of silk fiber substrate on the transformation of M1 macrophages to M2 macrophages was studied by inducing M0 macrophages to M1 macrophages. The specific experimental steps are as follows: (1) When RAW267.4 cells were cultured in a culture flask to 70-80% confluence, the cells were plated at 5×10 4 Cells / mL were seeded in a twelve-well plate, with three parallel wells set up in each well. (2) After 12 hours, the culture medium was aspirated and washed three times with sterile PBS. 500 μL of 0.5 μg / mL lipopolysaccharide (LPS) (prepared in DMEM) was added to each well for induction for 12 hours. The culture medium was precipitated and washed 2-3 times with PBS. 1 mL of complete DMEM culture medium was added and placed on each group of silk fiber substrates and incubated for 48 hours. The culture medium was discarded and washed three times with PBS. (3) At room temperature, 500 μL of 4% paraformaldehyde was added to each well and incubated for 10 minutes to fix the cells. The paraformaldehyde was aspirated and washed three times with PBS. (4) 500 μL of 0.1% Trixton-100 was added to each well and permeabilized for 10 minutes at room temperature. The permeabilization agent was removed and the wells were washed three times with PBS, each for 5 minutes. (5) 1 mL of 1% BSA solution was added to each well and incubated for 1 hour at room temperature to block the nonspecific binding of the antibody. (6) Add 500 μL of diluted primary antibody (M1 type primary antibody is iNOS, M2 type primary antibody is MRC-1) to each well and incubate overnight at 4°C. (7) Aspirate the primary antibody and wash the cells with PBS three times, 5 minutes each time. (8) Add 500 μL of diluted secondary antibody (dissolved in PBS containing 1% BSA) to each well and incubate at 37°C in the dark for 2 hours. (9) Aspirate the secondary antibody and wash the cells with PBS three times, 5 minutes each time in the dark. (10) Add 500 μL of DAPI staining solution to each well and counterstain the cell nuclei for 10 minutes. (11) Observe the fluorescence intensity using an ultra-high resolution confocal microscope.

[0083] The results of macrophage polarization experiments were as follows Figure 9As shown in the figure, compared with the control group, the red fluorescence of the THFSC group was weakened, while that of the TH-PDA and TH-PDA@Mg2 groups was significantly weakened. Meanwhile, the green fluorescence of the THFSC group was enhanced, while that of the TH-PDA and TH-PDA@Mg2 groups was significantly enhanced. This indicates that after the silk fiber substrate is treated with the ternary solution, THFSC has low immunogenicity due to the partial dissolution of sericin, and can induce macrophage phenotypic transformation. The addition of magnesium ions further facilitates the phenotypic transformation of macrophages.

[0084] Polarization experiments showed that the silk fiber substrate with polydopamine grafted with magnesium ions can promote the phenotypic transformation of macrophages.

[0085] Test Example 4 Alizarin Red and Alkaline Phosphatase Staining Experiment

[0086] Alkaline phosphatase (ALP) is an essential enzyme for bone formation and an early marker of osteoblast differentiation and functional maturation. To evaluate the effects of each silk fiber substrate on Mc3t3-e1 osteogenic differentiation, alkaline phosphatase staining was used to measure its expression. Mineralized calcium nodules are a hallmark of osteoblast differentiation and maturation and are the primary morphological characteristic of osteoblasts in their osteogenesis. Observing mineralized calcium nodules in osteoblasts is a commonly used technique for studying osteoblast differentiation.

[0087] The results of the alizarin red and alkaline phosphatase staining experiments are as follows Figure 10 Alizarin red and alkaline phosphatase staining experiments showed that the surface-treated hot-pressed silk fiber matrix grafted with magnesium ions using polydopamine solution had excellent osteogenic ability.

[0088] Test Example 5 Animal Experiment

[0089] The animal experiment used in this embodiment is male SD rats (6-8 weeks old, weighing 200 ± 20g), and 30 rats are divided into three groups: blank control group, THFSC, and TH-PDA@Mg2, with 5 rats in each group. Each rat is anesthetized with ether. The specific method is: a sterile cotton ball or gauze soaked with ether is placed in a beaker (1000ml), the experimental animal is placed in it, sealed with plastic film, and observed. It will be found that the animal first starts to get excited, then becomes inhibited, and falls down on its own. Because the experimental time is too long, the animal can be fixed on the experimental table, and the cotton ball containing ether is placed close to its nose to maintain the inhalation state. After the onset, the skull area is prepared, disinfected, draped, and the rat is placed in a prone position. A 3cm longitudinal incision is made in the rat skull, the skin, subcutaneous tissue and muscle-periosteum layer are cut open, and the skull bone plate is exposed by blunt separation. A full-thickness periosteal bone defect with a diameter of 8mm is made in the skull suture with a small dental drill. In the blank control group, no material was placed, and the subcutaneous layer was directly sutured with 5-0 silk suture. After disinfection, the skin layer was sutured with 1# silk suture. In the THFSC silk scaffold group and the PDA@Mg composite silk scaffold group, the material was placed before the skin was sutured. Rats were kept warm until they regained consciousness and housed in separate cages. 200,000 units of penicillin were injected intraperitoneally daily for three consecutive days. Rats were weighed and their health was monitored every three days.

[0090] Six weeks after surgery, rats were anesthetized by inhalation of an overdose of ether gas (same method as above) and then given animal-assisted cervical dislocation. The rat skulls were removed and fixed in 4% paraformaldehyde solution. The skulls were scanned and analyzed using Micro-CT. The obtained Micro-CT three-dimensional reconstruction images are shown in Figure 2. Figure 11 As shown by Figure 11 It can be seen that 6 weeks after implantation of the scaffold material, new bone formation was observed at the defect edges of both the THFSC and TH-PDA@Mg2 treatment groups, and more new bone was generated in the TH-PDA@Mg2 treatment group, while almost no new bone formation was observed in the Control group.

[0091] At the same time, the bone volume fraction (BV / TV) was quantified 6 weeks after surgery. The quantitative results were as follows: Figure 12 As shown in the figure, the results showed that TH-PDA@Mg2 (12.17±1.06%) had more mineralized matrix bone formation than THFSC (9.43±0.92%) and Control (7.58±1.15%). This indicates that the TH-PDA@Mg2 group has a better effect on bone repair and a better repair effect.

[0092] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a silk scaffold material modified with polydopamine and magnesium ions, comprising the following steps: The 0.1-0.3 cm flat silk is stacked into a multi-layer flat silk with a thickness of 2-4 layers, and the multi-layer flat silk is thermoplastically formed using a hot press, wherein the hot pressing temperature is 105° C., the time is 1 hour, and the pressure is 100 MPa to form a hot-pressed silk fiber substrate; The hot-pressed silk fiber substrate was pretreated in a ternary solution with a molar ratio of CaCl2:EtOH:H2O of 1:2:8, and the immersion temperature was 80°C for 10 minutes. The pretreated hot-pressed silk fiber substrate was placed in a refrigerator and frozen at -80°C for 12 hours, followed by vacuum freeze drying to form a surface-treated hot-pressed silk fiber substrate. Use 10mL of 10mM, pH=8.5 Tris-HCl buffer as a solvent to dissolve 20mg of dopamine hydrochloride to obtain a polydopamine solution; soak the surface-treated hot-pressed silk fiber substrate in the polydopamine solution, add MgSO4, and control the concentration of MgSO4 in the polydopamine solution to 0.0001mol / L. After 24 hours, take out the material and wash it three times with deionized water to form a silk scaffold material modified with polydopamine and magnesium ions.

2. The polydopamine and magnesium ion modified silk scaffold material prepared by the preparation method of claim 1 comprises a hot-pressed silk fiber substrate and polydopamine and magnesium ions modified on the surface of the silk fiber substrate.

3. Use of the polydopamine and magnesium ion modified silk scaffold material according to claim 2 in the preparation of bone repair materials.

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