CN116850346A - Polydopamine and magnesium ion modified silk scaffold material and preparation method and application thereof - Google Patents

Abstract

The invention provides a polydopamine and magnesium ion-modified silk scaffold material and its preparation method and application, and belongs to the technical field of bone repair materials. The invention uses hot-pressed flat silk as the base material, eliminating a series of tedious steps such as cocoon cutting and degumming in traditional techniques. It is simple to operate, low-cost, and the process is environmentally friendly. After hot-pressing the flat silk, the mechanics of the silk support material are improved. The performance is greatly improved; the present invention uses a ternary solution to micro-process the silk fiber base material to increase the surface roughness of the base material, which is beneficial to the subsequent adhesion and growth of cells, and then polydopamine formed by dopamine self-polymerization is grafted on the surface of the material. Improve the hydrophilicity and biocompatibility of the silk fiber base material; magnesium ions can replace the hydroxyl groups in the catechol structure of polydopamine, and use the grafting effect of polydopamine to graft the magnesium ions onto the surface of the silk fiber base material, thereby It has excellent effects in regulating macrophage polarization, promoting angiogenesis and promoting bone regeneration.

Description

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

Technical field

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 and its preparation method and application.

Background technique

The incidence of bone injuries from accidents and illness is increasing worldwide. In addition, as the world's population continues to grow, the proportion of the elderly population continues to increase, which has led to a significant increase in the incidence of orthopedic diseases, and subsequently the demand and difficulty of bone repair have also increased significantly. Biomaterials currently used for bone repair are divided into the following categories: medical bioceramics, medical polymer materials, medical composite materials, and nano-artificial bone.

Medical bioceramic materials mainly use hydroxyapatite (HAP) to repair bone tissue. Its composition and properties are very similar to HAP in biological hard tissue. It also has good biocompatibility and can form a strong bone with natural bone. Bonding is beneficial to subsequent mineralization and bone tissue regeneration; however, its strength is low and it is not suitable as a load-bearing component of the human body.

High molecular polymers have been widely used as bone repair materials, among which degradable polylactic acid (PLA) is used in oral surgery, polymethylmethacrylate (PMMA) bone cement is used for bone filling, and polyglycolic acid (PGA) is used as a biodegradable bone repair material. Absorbable screws are used for bone fixation. The elastic modulus of bone grafting materials made of biodegradable materials is closer to that of bone tissue than metals. As the fracture heals, the material gradually degrades in the body, eliminating the need for secondary surgery to remove it.

Silk, as a natural polymer biomaterial, has established a good 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 of silk fibroin, silk fibroin can be processed into various biological materials such as silk fibroin porous sponges, silk fibroin hydrogels, silk fibroin-based scaffolds, and silk fibroin nanoparticles. However, the traditional application of silk protein requires a series of processing of silk. The process is complex, the cost is increased, and it is easy to pollute the environment, so it is difficult to achieve widespread application.

Contents of the invention

In view of this, the object of the present invention is to provide a polydopamine and magnesium ion-modified silk scaffold material and its preparation method and application. The preparation method provided by the invention is simple to operate, low in cost, and the process is environmentally friendly. The obtained silk scaffold material has excellent mechanical properties and biocompatibility, and can regulate macrophage polarization, promote angiogenesis, and promote bone regeneration.

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

The invention provides a method for preparing a polydopamine- and magnesium-ion-modified silk scaffold material, which includes the following steps:

(1) Hot-press the stacked multi-layer flat silk to obtain a hot-pressed silk fiber base material;

(2) Soak the hot-pressed silk fiber base material in a ternary solution, take it out and freeze-dry it to obtain a pretreated silk fiber base material; the ternary solution includes CaCl ₂ , ethanol and water;

(3) The pretreated silk fiber base material is soaked in a polydopamine solution containing magnesium ions and modified to obtain a polydopamine- and magnesium ion-modified silk scaffold material.

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

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

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

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

Preferably, the preparation method of the polydopamine solution containing magnesium ions includes the following steps:

Mix dopamine hydrochloride and Tris-HCl solution to perform a self-polymerization reaction to obtain a polydopamine solution;

The soluble magnesium salt is mixed with the polydopamine solution to obtain a polydopamine solution containing magnesium ions.

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

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

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

The invention provides a polydopamine- and magnesium-ion-modified silk scaffold material prepared by the above preparation method, including a hot-pressed silk fiber base material and polydopamine and magnesium ions modified on the surface of the silk fiber base material.

The invention provides the application of the above-mentioned polydopamine and magnesium ion-modified silk scaffold materials in the preparation of bone repair materials.

The invention provides a method for preparing a polydopamine- and magnesium-ion-modified silk scaffold material, which includes the following steps: (1) hot-pressing stacked multi-layer flat-plate silks to obtain a hot-pressed silk fiber base material; (2) The hot-pressed silk fiber base material is soaked in a ternary solution, taken out and then freeze-dried to obtain a pretreated silk fiber base material; the ternary solution includes CaCl ₂ , ethanol and water; (3) the pretreatment The silk fiber base material is soaked in a polydopamine solution containing magnesium ions and modified to obtain a polydopamine- and magnesium ion-modified silk scaffold material. The invention uses hot-pressed flat silk as the base material, eliminating a series of tedious steps such as cocoon cutting and degumming in traditional techniques. It is simple to operate, low-cost, and the process is environmentally friendly. After hot-pressing the flat silk, the mechanics of the silk support material are improved. The performance is greatly improved; the present invention uses a ternary solution to micro-process the silk fiber base material to increase the surface roughness of the base material, which is beneficial to the subsequent adhesion and growth of cells. The polydopamine formed by dopamine self-polymerization relies on its own sticky protein The structure is grafted on the surface of the material to improve the hydrophilicity of the silk fiber base material and make it have better biocompatibility; magnesium ions can replace the hydroxyl groups in the catechol structure of polydopamine. The present invention utilizes the grafting of polydopamine. The magnesium ions are grafted onto the surface of the silk fiber substrate through dendritic effects, thereby exerting excellent effects in regulating macrophage polarization, promoting angiogenesis and promoting bone regeneration. The Mc3t3-e1 cell and macrophage proliferation experiment shows that the biocompatibility of the silk fiber substrate is improved after cross-linking polydopamine; the polarization experiment shows that the polydopamine and magnesium ion-modified silk scaffold material of the present invention can promote the growth of macrophages. Phenotypic transformation; Alizarin red and alkaline phosphatase staining experiments show that the polydopamine and magnesium ion-modified silk scaffold material of the present invention has excellent osteogenic ability.

Description of the drawings

Figure 1 is a physical picture of hot-pressed silk fiber substrates with different treatments;

Figure 2 shows the infrared spectra of hot-pressed silk fiber substrates with different treatments;

Figure 3 shows the contact angles of hot-pressed silk fiber substrates with different treatments;

Figure 4 shows the stress-strain change diagram of hot-pressed silk fiber substrates with different treatments;

Figure 5 shows the EDS elemental analysis diagram of hot-pressed silk fiber substrates with different treatments;

Figure 6 is a quantitative chart of magnesium elements in hot-pressed silk fiber substrates with different treatments;

Figure 7 shows the proliferation results of Mc3t3-e1 cells;

Figure 8 shows the Raw cell proliferation results;

Figure 9 shows the results of macrophage polarization;

Figure 10 shows the experimental results of alizarin red and alkaline phosphatase staining;

Figure 11 shows the Micro-CT three-dimensional reconstructed image results;

Figure 12 shows the quantitative results of mouse bone volume fraction 6 weeks after surgery.

Detailed ways

The invention provides a method for preparing a polydopamine- and magnesium-ion-modified silk scaffold material, which includes the following steps:

(1) Hot-press the stacked multi-layer flat silk to obtain a hot-pressed silk fiber base material;

(2) Soak the hot-pressed silk fiber base material in a ternary solution, take it out and freeze-dry it to obtain a pretreated silk fiber base material; the ternary solution includes CaCl ₂ , ethanol and water;

(3) The pretreated silk fiber base material is soaked in a polydopamine solution containing magnesium ions and modified to obtain a polydopamine- and magnesium ion-modified silk scaffold material.

In the present invention, stacked multi-layer flat silks are hot-pressed to obtain a hot-pressed silk fiber base material. In the present invention, the flat silk is preferably a flat silk produced by five-instar mature silkworms spinning on a two-dimensional spinning bed plane. In the present invention, the weight of the flat yarn is preferably 2 to 3 g.

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

In the present invention, a hot press is preferably used to perform the hot pressing; in the present invention, the temperature of the hot pressing is preferably 80 to 130°C, more preferably 100 to 120°C; the hot pressing pressure is preferably 10 to 500MPa, more preferably It is 50-300MPa, more preferably 100-200MPa; the temperature and pressure holding time is preferably 5-180min, more preferably 10-150min, further preferably 30-100min. In the present invention, the silk fiber base material is thermoplastically formed through the hot pressing.

After obtaining the hot-pressed silk fiber base material, the present invention soaks the hot-pressed silk fiber base material in a ternary solution, takes it out and freeze-dries it to obtain a pretreated silk fiber base material; the ternary solution includes CaCl ₂ , ethanol and water. In the present invention, in the ternary solution, the molar ratio of CaCl ₂ , ethanol and water is preferably 0.9-1:2:6.5-8, and more preferably 1:2:8, 1:2:7, 1: 2:6.5 or 0.9:2:8.

In the present invention, the soaking temperature is preferably 60-100°C, more preferably 70-80°C; the soaking time is preferably 5-15 min, more preferably 10 min. The present invention uses a suitable ternary solution to micro-treat the surface of the material for a period of time, so that part of the sericin on the surface of the material is dissolved in the ternary solution, causing the material surface to be uneven and irregular, which to a certain extent increases the roughness of the surface of the hot-pressed silk fiber base material. Spend.

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 mode of the vacuum freeze-drying, and it is enough to use the vacuum freeze-drying mode well known to those skilled in the art.

After obtaining the pretreated silk fiber base material, the present invention soaks the pretreated silk fiber base material in a polydopamine solution containing magnesium ions and performs modification to obtain a polydopamine and magnesium ion modified silk scaffold material. In the present invention, the preparation method of the polydopamine solution containing magnesium ions preferably includes the following steps:

Mix dopamine hydrochloride and Tris-HCl solution to perform a self-polymerization reaction to obtain a polydopamine solution;

The soluble magnesium salt is mixed with the polydopamine solution to obtain a polydopamine solution containing magnesium ions.

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, and more preferably 16 to 20 hours.

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

In the present invention, the soluble magnesium salt is preferably MgSO ₄ , and the concentration of magnesium ions in the polydopamine solution containing magnesium ions is preferably 0.00005 to 0.01 mol/L, and more preferably 0.0001 to 0.0004 mol/L.

In the present invention, the order in which the pretreated silk fiber base material is soaked in the polydopamine solution containing magnesium ions is preferably: first soak the pretreated silk fiber base material in the polydopamine solution, and then add soluble magnesium salt.

In the present invention, the temperature of the modification is preferably room temperature, and the time is preferably 12 to 24 hours, and more preferably 16 to 20 hours. In the present invention, during the modification process, polydopamine is grafted on the surface of the pretreated silk fiber base material, and magnesium ions replace the hydroxyl groups in the catechol structure of dopamine, thereby realizing the modification of magnesium ions on the surface of the silk fiber base material.

After the modification, in the present invention, 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.

The invention provides a polydopamine- and magnesium-ion-modified silk scaffold material prepared by the above preparation method, including a hot-pressed silk fiber base material and polydopamine and magnesium ions modified on the surface of the silk fiber base material.

The invention provides the application of the above-mentioned polydopamine and magnesium ion-modified silk scaffold materials in the preparation of bone repair materials. The polydopamine and magnesium ion-modified silk scaffold material provided by the invention has excellent mechanical properties and biocompatibility, can regulate macrophage polarization, promote angiogenesis and promote bone regeneration, and can be used as a treatment for bone defects in different parts of the body. Restorative scaffold materials.

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

Example 1

A certain number of fifth-instar mature silkworms are placed on a two-dimensional spinning bed plane to spin silk to obtain flat silk of 0.1 to 0.3 cm. The flat silk obtained is stacked into a multi-layered flat plate with a thickness of 2 to 4 layers. Silk, use a hot press to thermoform multiple layers of flat silk. 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 base material.

The hot-pressed silk fiber substrate is pretreated in a ternary solution. The molar ratio of CaCl ₂ :EtOH:H ₂ O in the ternary solution is 1:2:8. The soaking temperature is 80°C and the time is 10 minutes. The pretreated hot-pressed silk fiber base material is placed in a refrigerator and frozen at a freezing temperature of -80°C for 12 hours, and then vacuum freeze-dried to form a surface-treated hot-pressed silk fiber base material.

Use 10mL Tris-HCl buffer (10mM, pH=8.5) as the 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 MgSO ₄ and control MgSO ₄ The concentration in the polydopamine solution was 0.00005 mol/L. After 24 hours, the material was taken out and washed three times with deionized water to form a polydopamine and magnesium ion-modified silk scaffold material, recorded as TH-PDA@Mg1.

Example 2

The difference from Example 1 is that the concentration of MgSO ₄ in the polydopamine solution is 0.0001 mol/L, which is recorded as TH-PDA@Mg2.

Example 3

The difference from Example 1 is that the concentration of MgSO ₄ in the polydopamine solution is 0.0002 mol/L, which is recorded as TH-PDA@Mg3.

Example 4

The difference from Example 1 is that the concentration of MgSO ₄ in the polydopamine solution is 0.0004 mol/L, which is recorded as TH-PDA@Mg4.

Example 5

The difference from Example 1 is that the concentration of MgSO ₄ in the polydopamine solution is 0.001 mol/L, which is recorded as TH-PDA@Mg5.

Test example 1

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

The physical pictures of hot-pressed silk fiber substrates with different treatments are shown in Figure 1, and the infrared spectra are shown in Figure 2. From the physical images and infrared spectra, it can be observed that dopamine successfully self-polymerized into polydopamine and was cross-linked on the surface of the silk fiber substrate.

The contact angles of hot-pressed silk fiber substrates with different treatments are shown in Figure 3, and the stress-strain change diagram is shown in Figure 4. The contact angle and stress-strain showed that the silk fiber substrate modified with polydopamine and magnesium ions 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.

EDS tests were conducted on hot-pressed silk fiber substrates with different treatments. The obtained EDS elemental analysis chart is shown in Figure 5. In Figure 5, red represents carbon, green represents nitrogen, blue represents oxygen, and yellow represents magnesium. The quantitative graph of magnesium element of hot-pressed silk fiber substrates with different treatments is shown in Figure 6.

It can be seen from Figures 5 and 6 that polydopamine was successfully used to graft magnesium ions on the surface of the silk fiber substrate, and as the concentration of magnesium ions increased, the magnesium ions on the surface of the silk fiber substrate also increased.

Test Example 2 Mc3t3-e1 Cell and Macrophage Proliferation Experiment

The Mc3t3-e1 cells used in this test example were purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences/Cell Resource Center of the Shanghai Academy of Biological Sciences, Chinese Academy of Sciences; the RAW264.7 cells were from this research group. Mc3t3-e1 was cultured in α-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% _CO2 , and the medium was changed every 3 days; RAW264.7 cells were cultured in α-MEM medium containing 10% penicillin-streptomycin at 37°C and 5% CO2. DMEM culture medium with fetal bovine serum and 1% penicillin and streptomycin was cultured at 37°C and 5% _CO2 , and the culture medium was replaced every 3 days. Soak the silk fiber substrates of each group in 75% alcohol for 2 to 3 hours, rinse with PBS 3 to 5 times, and then place them in a UV environment for 2 to 3 hours. Mc3t3-e1 cells and RAW264.7 were seeded in a 48-well plate at a density of 5×10 ⁴ Cell/well. After 24 hours, the above-mentioned sterilized materials were placed in the well plate and the cells were cultured in an incubator. Use the MTS method to detect the proliferative activity of the two cells. Take out the cell plate at the predetermined time point (24h48h), aspirate the culture medium, rinse slowly with PBS, add 500 μL MTS working solution to each well and place it in a cell culture incubator to avoid light and incubate for 2h. . Finally, 100 μL of the supernatant was pipetted into a 96-well plate, and the absorbance at a wavelength of 490 nm was detected by a microplate reader. Medium containing 10% FBS was inoculated as a control, and its absorbance value was used to determine 100% viability of the cells. All experimental groups were set up with three biological replicates. 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.

The Mc3t3-e1 cell proliferation results are shown in Figure 7, and the Raw cell proliferation results are shown in Figure 8. The results show that the biocompatibility of silk fiber substrates is improved after cross-linking polydopamine.

Test Example 3 Macrophage Polarization Experiment

Macrophages, as a precursor cell type that triggers innate immune responses, have unique functional and phenotypic characteristics. Macrophages can be divided into classically activated M1 phenotypes and alternatively activated M2 phenotypes based on their functions and surface markers. M1 macrophages are generally considered to express high levels of pro-inflammatory cytokines, such as nitric oxide enzyme (iNOS), vascular endothelial growth factor (VEGF), interleukin IL-6, tumor necrosis factor TNF-α, etc., which induce Inflammatory infiltration; M2 macrophages usually have the characteristics of anti-inflammatory molecules, such as mannose receptor MRC-1 (also known as CD206), chemokines, glutamine transaminase 2, etc. Macrophages gather at the implantation site and adhere to the implanted material to form giant cells, which secrete various chemical components to regulate the microenvironment and play a vital role in tissue regeneration. Studies have reported that the polarization of M2 macrophages induced by biomaterials is beneficial to the rapid repair of bone injury regeneration.

This test example studies the effect of silk fiber substrate on the transformation of M1 macrophages into M2 macrophages by inducing M0 macrophages into M1 macrophages. The specific experimental steps are as follows: (1) RAW267.4 is cultured in a culture bottle until When 70 to 80% confluence, the cells were seeded in a twelve-well plate at 5×10 ⁴ Cell/mL, and three parallel wells were set in each well. (2) After 12 hours, aspirate the culture medium, wash it 3 times with sterile PBS, add 500 μL 0.5 μg/mL lipopolysaccharide LPS (prepared in DMEM) to each well for induction for 12 hours, separate out the culture medium, wash it 2 to 3 times with PBS, and add 1 mL DMEM Complete culture medium was placed into each group of silk fiber substrates, and incubated for 48 hours. Discard the culture medium and wash 3 times with PBS. (3) At room temperature, add 500 μL of 4% paraformaldehyde to each well and incubate for 10 minutes to fix the cells. Aspirate the paraformaldehyde and wash 3 times with PBS. (4) Add 500 μL of 0.1% Trixton-100 to each well and permeabilize for 10 minutes at room temperature. Remove the permeabilizing agent and wash with PBS 3 times for 5 minutes each time. (5) Add 1 mL of 1% BSA solution to each well and incubate at room temperature for 1 hour to block non-specific binding of the antibody. (6) Add 500 μL of diluted primary antibody to each well (M1 type primary antibody is iNOS, M2 type primary antibody is MRC-1) and incubate overnight at 4°C. (7) Aspirate the primary antibody and wash the cells with PBS 3 times for 5 minutes each time. (8) Add 500 μL of diluted secondary antibody (dissolved in PBS containing 1% BSA) to each well and incubate for 2 hours at 37°C in the dark. (9) Aspirate the secondary antibody and wash 3 times with PBS in the dark, 5 minutes each time. (10) Add 500 μL DAPI dye solution to each well and counterstain cell nuclei for 10 min. (11) Use ultra-high-resolution confocal microscopy to observe fluorescence intensity.

The results of macrophage polarization experiments are shown in Figure 9. It can be seen that compared with the Control group, the red fluorescence of the THFSC group was weakened, while the red fluorescence of the TH-PDA group and TH-PDA@Mg2 group was greatly weakened; at the same time, the green fluorescence of the THFSC group was enhanced, while the TH-PDA group and TH-PDA group were significantly weakened. The green fluorescence of the @Mg2 group was greatly enhanced. It shows that after the silk fiber substrate is treated with the ternary solution, due to the partial dissolution of sericin, THFSC has low immunogenicity and can induce the phenotype transformation of macrophages. At the same time, the addition of magnesium ions can be more beneficial to the phenotype of macrophages. Transformation.

Polarization experiments show that polydopamine-grafted silk fiber substrates with magnesium ions can promote the phenotypic transformation of macrophages.

Test Example 4 Alizarin Red and Alkaline Phosphatase Staining Experiment

Alkaline phosphatase (ALP) is an essential enzyme for bone formation and an early marker of osteogenic differentiation and functional maturation. In order to evaluate the effect of each group of silk fiber substrates on Mc3t3-e1 osteogenic differentiation, alkaline phosphatase staining was used method to determine its expression activity. Mineralized calcium nodules are a sign of osteoblast differentiation and maturity, and are also the main morphological characteristics of osteoblasts performing osteogenic functions. Observing mineralized calcium nodules of osteoblasts is one of the common techniques for studying osteoblast differentiation.

The experimental results of alizarin red and alkaline phosphatase staining are shown in Figure 10. Alizarin red and alkaline phosphatase staining experiments show that the surface treatment of hot-pressed silk fiber base after grafting magnesium ions with polydopamine solution has excellent osteogenic ability.

Test Example 5 Animal Experiment

The animal experiments used in this example were male SD rats (6 to 8 weeks old, weight 200±20g). 30 rats were divided into three groups, blank control group, THFSC, TH-PDA@Mg2, each group had 5 rats. Only. Anesthetize each rat with ether. The specific method is as follows: put sterilized absorbent cotton balls or gauze soaked in ether into a beaker (1000ml), put the experimental animal in, seal it with a plastic film, observe, you will find that the animal first Begins to be excited, then becomes inhibited and collapses on his own. Since the experiment 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 of effect, the skull area was skinned, disinfected, draped, and the rat placed in a prone position. A 3cm longitudinal incision was made on the skull of the rat. The skin, subcutaneous tissue, and muscle-periosteal layer were incised and flapped. Blunt dissection was performed to expose the skull bones. plate. A small dental drill was used to create a full-thickness periosteal bone defect with a diameter of 8 mm in the midcranial suture of the skull. In the blank control group, no material was placed, and the subcutaneous layer was sutured directly with 5-0 silk thread. After disinfection, the skin layer was sutured with 1# silk thread. The THFSC silk scaffold group and PDA@Mg composite silk scaffold were put into the material and then the skin was sutured. After surgery, the rats were kept warm until they woke up and were raised in separate cages. After surgery, 200,000 units of penicillin were injected intraperitoneally every day for three consecutive days. The weight of the rats was measured every three days and the health status of the rats was observed.

Six weeks after the operation, the rats were anesthetized by inhaling excessive ether gas (the same method as above), and then the animals were given auxiliary neck dissection. The rat skull was removed and fixed and preserved in 4% paraformaldehyde solution. Skull scans were analyzed using Micro-CT. The obtained Micro-CT three-dimensional reconstructed image results are shown in Figure 11. It can be seen from Figure 11 that 6 weeks after implanting the scaffold material, new bone formation was observed at the edge of the defect in both the THFSC and TH-PDA@Mg2 treatment groups, and TH -The PDA@Mg2 treatment group produced more new bone, while the Control group produced almost no new bone.

At the same time, the bone volume fraction (BV/TV) was quantified 6 weeks after surgery. The quantitative results are shown in Figure 12. The results showed that TH-PDA@Mg2 (12.17±1.06%) was compared with THFSC (9.43±0.92%) and Control. (7.58±1.15%), there was more mineralized matrix bone formation. This shows that the TH-PDA@Mg2 group has better bone repair effect and better repair effect.

The above are only preferred embodiments of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principles of the present invention. These improvements and modifications can also be made. should be regarded as the protection scope of the present invention.

Claims (10)

1. A preparation method of a polydopamine and magnesium ion modified silk scaffold material comprises the following steps:

(1) Carrying out hot pressing on the stacked multi-layer flat silk to obtain a hot-pressed silk fiber base material;

(2) Soaking the hot-pressed silk fiber substrate in a ternary solution, taking out, and freeze-drying to obtain a pretreated silk fiber substrate; the ternary solution comprises CaCl ₂ Ethanol and water;

(3) And soaking the pretreated silk fiber base material in a polydopamine solution containing magnesium ions for modification to obtain the polydopamine and magnesium ion modified silk scaffold material.

2. The method of claim 1, wherein the multilayer flat wire has a stack thickness of 0.1 to 30cm;

the hot pressing temperature is 80-130 ℃, the hot pressing pressure is 10-500 MPa, and the heat preservation and pressure maintaining time is 5-180 min.

3. The method of claim 1, wherein CaCl is present in the ternary solution ₂ The mol ratio of ethanol to water is 0.9-1:2:6.5-8.

4. A method according to claim 1 or 3, wherein the soaking temperature in step (2) is 60 to 100 ℃ for 5 to 15 minutes.

5. The preparation method according to claim 1, wherein the preparation method of the polydopamine solution containing magnesium ions comprises the following steps:

mixing dopamine hydrochloride with Tris-HCl solution, and performing self-polymerization reaction to obtain polydopamine solution;

and mixing the soluble magnesium salt with the polydopamine solution to obtain the polydopamine solution containing magnesium ions.

6. The method according to claim 5, wherein the Tris-HCl solution has a concentration of 10mM and a pH of 8.5.

7. The method according to claim 1 or 5, wherein the concentration of magnesium ions in the polydopamine solution is 0.00005 to 0.01mol/L and the concentration of polydopamine is 2 to 4mg/mL.

8. The method according to claim 1, wherein the modification time in the step (3) is 12 to 24 hours.

9. The polydopamine and magnesium ion modified silk scaffold material prepared by the preparation method according to any one of claims 1-8, which comprises a hot-pressed silk fiber substrate and polydopamine and magnesium ions modified on the surface of the silk fiber substrate.

10. Use of polydopamine and magnesium ion modified silk scaffold material according to claim 9 for the preparation of bone repair material.