Preparation method of conductive multi-layer composite membrane for promoting bone regeneration

Abstract

The invention relates to the technical field of biological functional polymer materials, and discloses a preparation method of a conductive multi-layer composite membrane for promoting bone regeneration, which comprises the following steps: firstly, adding platelet-rich fibrin powder into a silk fibroin aqueous solution, and fully and uniformly mixing; then, preparing a dispersion liquid of MXene of which the surface is loaded with the strontium and selenium-containing silk fibroin nanofibers; then alternately spin-coating the silk fibroin / platelet-rich fibrous protein mixed solution and the MXene dispersion liquid of which the surface is loaded with the strontium and selenium-containing silk fibroin nanofibers on a silicon wafer with a cellulose acetate layer through a spin-coating layer-by-layer technology, and repeating for multiple times to obtain a composite film with a certain thickness; and finally, stripping the composite film from the silicon wafer, and carrying out water vapor treatment on the composite film. The composite membrane has electrical conductivity, blood compatibility, osteoinductivity and long-acting stable delivery capacity, and shows good clinical application potential in the fields of bone tissue engineering and regenerative medicine.

Description

A method for preparing a conductive, bone-regenerating multilayer composite membrane Technical Field

[0001] This invention relates to the field of biofunctional polymer materials technology, and in particular to a method for preparing a conductive, bone-regenerating multilayer composite membrane. Background Technology

[0002] Bone repair is a complex physiological process synergistically regulated by multiple biochemical and physical factors in the microenvironment, requiring precise design and dynamic control. Bone tissue is electrosensitive and responds significantly to endogenous bioelectrical signals, while bone defects often lead to the loss of these signals. Therefore, developing conductive scaffold materials to supplement these signals and promote bone regeneration has become one of the key research directions.

[0003] Silk fibroin is considered an ideal matrix material for bone regeneration scaffolds due to its excellent biocompatibility, tunable degradability, and mechanical strength. However, its intrinsic non-conductivity limits its application in electroactive bone repair. To impart conductivity, current techniques often introduce conductive nanoparticles into the silk fibroin matrix through blending. However, the poor interfacial compatibility between nanoparticles and the matrix easily leads to aggregation, thereby weakening the mechanical properties of the composite material.

[0004] In addition, bioactive ions such as strontium (Sr) 2+ ) and selenium (Se) 2+ Osteoblasts play a crucial role in bone regeneration, promoting both osteoblast proliferation and differentiation. However, their release rate requires precise control: too rapid a release leads to burst release, while too slow release results in insufficient biological activity, both affecting osteogenic efficacy. Therefore, achieving controllable ion release is one of the core challenges in endowing scaffolds with osteoinductive function.

[0005] In summary, how to synergistically address the dispersion problem of nanoparticles and achieve the controllable release of strontium and selenium ions has become a key scientific issue in constructing functional conductive bone repair materials. However, existing research mostly focuses on optimizing single performance aspects and lacks a systematic strategy that organically integrates conductive scaffold construction with active ion delivery systems. Summary of the Invention

[0006] Purpose of the invention: To address the problems existing in the prior art, this invention provides a method for preparing a conductive, bone-regenerating multilayer composite membrane that can simultaneously control the release of strontium and selenium ions and impart conductivity and excellent mechanical properties.

[0007] Technical solution: In the first aspect, the present invention provides a method for preparing a conductive multilayer composite membrane for promoting bone regeneration, comprising the following steps: uniformly mixing a silk fibroin aqueous solution with platelet-rich fibrin to obtain a silk fibroin / platelet-rich fibrin mixed aqueous solution; preparing a dispersion of MXene containing strontium and selenium-containing silk fibroin nanofibers loaded on the surface; alternately spin-coating the silk fibroin / platelet-rich fibrin mixed aqueous solution and the dispersion of MXene containing strontium and selenium-containing silk fibroin nanofibers loaded on the surface onto a silicon wafer with a cellulose acetate layer by alternating spin-coating technology, repeating the process multiple times to form a composite membrane of a certain thickness; placing the silicon wafer obtained in step (3) in acetone to dissolve and remove the cellulose acetate layer, peeling the composite membrane off the silicon wafer, drying it, and then treating the composite membrane with water vapor to obtain a conductive multilayer composite membrane for promoting bone regeneration.

[0008] Further, in step (1), platelet-rich fibrin powder is added to a silk fibroin aqueous solution with a mass fraction of 2-6% in a certain proportion, and the mixture is stirred slightly to make the two evenly mixed to obtain a silk fibroin / platelet-rich fibrin mixed aqueous solution; wherein, the mass ratio of platelet-rich fibrin powder to silk fibroin is 0.01-5:100.

[0009] Further, in step (2), the specific preparation process of the MXene dispersion with strontium and selenium-containing silk fibroin nanofibers loaded on the surface is as follows: S1, dissolve sodium selenite and strontium chloride in a silk fibroin aqueous solution with a mass fraction of 4-8%; wherein, the concentrations of sodium selenite and strontium chloride in the mixed solution are 0.01-5 mol / L and 0.01-5 mol / L, respectively. S2. After ultrasonically dispersing the monolayer MXene in water, add the mixture obtained in S1 to make the mass ratio of MXene to silk fibroin 0.01-1:10, and mix gently and evenly; S3. Concentrate the mixture obtained in S2 at 60℃. When the concentration of silk fibroin in the mixture is 20-30%, take it out and add deionized water to dilute it so that the mass fraction of silk fibroin in the mixture is reduced to 1-4%. Then, treat it again at 60℃ for 24 hours; S4. Centrifuge the mixture obtained in S3, remove the supernatant, and obtain MXene with strontium and selenium-containing silk fibroin nanofibers on the surface. After freeze-drying, disperse it in methanol to obtain a dispersion of MXene with strontium and selenium-containing silk fibroin nanofibers on the surface with a concentration of 0.1-1%.

[0010] Further, in step (3), the specific conditions for a single spin coating of the silk fibroin / platelet-rich fibrin mixed aqueous solution are: spin coating speed of 2000-5000 rpm and time of 10-60 s; the specific conditions for a single spin coating of the dispersion of MXene containing strontium and selenium silk fibroin nanofibers loaded on the surface are: spin coating speed of 1000-3000 rpm and time of 10-20 s; the thickness of the composite film is 1-50 μm, and the outermost layer of the composite film is MXene particles of silk fibroin, strontium and selenium; the thickness of the cellulose acetate layer is 100-500 nm.

[0011] Furthermore, in step (4), the silicon wafer is placed in acetone and the immersion treatment time is 0.1-2 hours.

[0012] Furthermore, in step (4), the conditions for the water vapor treatment are: temperature of 30-40℃, humidity of 60-90%, and treatment time of 24-36 hours.

[0013] In a second aspect, the present invention provides the application of a conductive, bone-regenerating multilayer composite membrane prepared by any of the methods described above in bone repair.

[0014] Beneficial Effects: Compared with existing technologies, the specific beneficial effects of this invention are as follows: This invention achieves a systematic breakthrough in material performance through multi-level functionalized design: By utilizing spin-coating-assisted layer-by-layer assembly technology, a hierarchical multi-layer structure is constructed, which can effectively avoid performance degradation caused by poor nanoparticle dispersion. Functionally, the composite membrane prepared by this invention integrates the structural properties of silk fibroin and the bioactivity of platelet-rich fibrin. Furthermore, constructing silk fibroin nanofibers on the MXene surface and loading selenium (Se) and strontium (Sr) particles is a functionalized design, the core advantages of which lie in structural complementarity, functional synergy, and intelligent response. Specifically, MXene provides physical support and photoelectric response capabilities, while silk fibroin nanofibers, as biocompatible carriers and sustained-release platforms, enhance the structural stability of MXene while controllably promoting ion release; the release of Se / Sr dual elements synergistically exerts precise biochemical regulation effects of antioxidation and osteoproliferation. This system not only overcomes the performance limitations of single materials, but also has a simple and mild preparation process. The resulting composite membrane has conductivity, blood compatibility, osteoinductive properties, and long-term stable delivery capability, showing significant clinical application potential in the fields of bone tissue engineering and regenerative medicine. Attached Figure Description

[0015] Figure 1 shows the SEM images of MXene (a), MXene with strontium and selenium-containing silk fibroin nanofibers loaded on its surface (b), MXene with strontium and selenium-containing silk fibroin nanofibers loaded on its surface (c), and FTIR spectra of MXene and MXene with strontium and selenium-containing silk fibroin nanofibers loaded on their surface (d). Figure 2 shows the CCK-8 results of MC3T3-E1 osteoblasts co-cultured with samples from Examples 1-3 and Comparative Examples 1-4 for 3 days. Figure 3 shows the CCK-8 results of MC3T3-E1 osteoblasts co-cultured with Sample 1 of Example 1 for 3 days after exposure to 808 nm near-infrared (NIR) laser for 5 min without and after exposure to 808 nm NIR laser for 5 min. Figure 4 shows the growth morphology of MC3T3-E1 osteoblasts co-cultured with different samples for 3 days: (a) Comparative Example 2, (b) Comparative Example 3, (c) Comparative Example 4, (d) Example 1. (e) Example 1 (irradiation with 808nm laser for 5min), (f) Example 3; Figure 5 is a schematic diagram of the structure of the conductive bone regeneration-promoting multilayer composite membrane prepared in this invention. Detailed Implementation

[0016] The present invention will now be described in detail with reference to the embodiments. Example 1

[0017] 1. Add platelet-rich fibrin powder to a 2% (w / w) silk fibroin aqueous solution, wherein the mass ratio of platelet-rich fibrin to silk fibroin is 0.5:100. Stir gently to mix the two evenly to obtain a silk fibroin / platelet-rich fibrin mixed aqueous solution.

[0018] 2. Sodium selenite and strontium chloride were dissolved in a 4% (w / w) aqueous solution of silk fibroin, with concentrations of 1 mol / L for sodium selenite and 1 mol / L for strontium chloride. A monolayer of MXene was ultrasonically dispersed in water to obtain a homogeneous dispersion. This dispersion was added to the above solution, resulting in a MXene to silk fibroin mass ratio of 0.1:10. The resulting mixture was then concentrated at 60°C until the silk fibroin concentration reached 20%. Deionized water was then added to reduce the silk fibroin mass fraction to 1%, and the mixture was again treated at 60°C for 24 hours. The mixture was then removed, centrifuged, and the supernatant was discarded to obtain MXene with strontium and selenium-containing silk fibroin nanofibers loaded on its surface. After lyophilization, the MXene was dispersed in methanol to obtain a 0.1% (w / w) dispersion of MXene with strontium and selenium-containing silk fibroin nanofibers loaded on its surface.

[0019] 3. Using alternating spin-coating technology, a mixed aqueous solution of silk fibroin / platelet-rich fibrin and a dispersion of MXene containing strontium and selenium-containing silk fibroin nanofibers were alternately spin-coated onto a silicon wafer with a cellulose acetate layer (100 nm thick). Specifically, the silk fibroin / platelet-rich fibrin mixed aqueous solution was spin-coated at 2000 rpm for 10 s; the dispersion of MXene containing strontium and selenium-containing silk fibroin nanofibers was spin-coated at 1000 rpm for 10 s. Alternating spin-coating continued until the composite coating thickness reached 5 μm, with the outermost layer of the composite film consisting of silk fibroin, strontium, and selenium-containing MXene particles.

[0020] 4. Immerse the silicon wafer from step 3 in acetone for 20 minutes, then peel the composite film off the silicon wafer. After drying, place the composite film in a 30°C, 60% humidity environment for 24 hours. A conductive, bone-regenerating multilayer composite film is obtained. Example 2

[0021] 1. Add platelet-rich fibrin powder to a 4% (w / w) silk fibroin aqueous solution, wherein the mass ratio of platelet-rich fibrin to silk fibroin is 2:100. Stir gently to mix the two evenly to obtain a silk fibroin / platelet-rich fibrin mixed aqueous solution.

[0022] 2. Sodium selenite and strontium chloride were dissolved in a 6% (w / w) aqueous solution of silk fibroin, with concentrations of 2 mol / L for sodium selenite and 2 mol / L for strontium chloride. A monolayer of MXene was ultrasonically dispersed in water to obtain a homogeneous dispersion. This dispersion was added to the above solution, resulting in a MXene to silk fibroin mass ratio of 0.5:10. The resulting mixture was then concentrated at 60°C until the silk fibroin concentration reached 24%. Deionized water was then added to reduce the silk fibroin mass fraction to 2%, and the mixture was again treated at 60°C for 24 hours. The mixture was then removed, centrifuged, and the supernatant was discarded to obtain MXene with strontium and selenium-containing silk fibroin nanofibers loaded on its surface. After lyophilization, the MXene was dispersed in methanol to obtain a 0.25% (w / w) dispersion of MXene with strontium and selenium-containing silk fibroin nanofibers loaded on its surface.

[0023] 3. Using alternating spin-coating technology, a mixed aqueous solution of silk fibroin / platelet-rich fibrin and a dispersion of MXene containing strontium and selenium-containing silk fibroin nanofibers were alternately spin-coated onto a silicon wafer with a cellulose acetate layer (200 nm thick). Specifically, the silk fibroin / platelet-rich fibrin mixed aqueous solution was spin-coated at 3000 rpm for 25 s; the dispersion of MXene containing strontium and selenium-containing silk fibroin nanofibers was spin-coated at 1500 rpm for 15 s. Alternating spin-coating continued until the composite coating thickness reached 20 μm, with the outermost layer of the composite film consisting of silk fibroin, strontium, and selenium-containing MXene particles.

[0024] 4. Immerse the silicon wafer from step 3 in acetone for 40 minutes, then peel the composite film off the silicon wafer. After drying, place the composite film at 35°C and 75% humidity for 30 hours. A conductive, bone-regenerating multilayer composite film is obtained. Example 3

[0025] 1. Add platelet-rich fibrin powder to a 6% (w / w) silk fibroin aqueous solution, wherein the mass ratio of platelet-rich fibrin to silk fibroin is 4:100. Stir gently to mix the two evenly to obtain a silk fibroin / platelet-rich fibrin mixed aqueous solution.

[0026] 2. Sodium selenite and strontium chloride were dissolved in a 6% (w / w) aqueous solution of silk fibroin, with concentrations of 4 mol / L for sodium selenite and 4 mol / L for strontium chloride. A monolayer of MXene was ultrasonically dispersed in water to obtain a homogeneous dispersion. This dispersion was added to the above solution, resulting in a MXene to silk fibroin mass ratio of 1:10. The resulting mixture was then concentrated at 60°C until the silk fibroin concentration reached 30%. Deionized water was then added to reduce the silk fibroin mass fraction to 4%, and the mixture was again treated at 60°C for 24 hours. The mixture was then removed, centrifuged, and the supernatant was discarded to obtain MXene with strontium and selenium-containing silk fibroin nanofibers loaded on its surface. After lyophilization, the MXene was dispersed in methanol to obtain a 1% (w / w) dispersion of MXene with strontium and selenium-containing silk fibroin nanofibers loaded on its surface.

[0027] 3. Using alternating spin-coating technology, a mixed aqueous solution of silk fibroin / platelet-rich fibrin and a dispersion of MXene containing strontium and selenium-containing silk fibroin nanofibers were alternately spin-coated onto a silicon wafer with a cellulose acetate layer (500 nm thick). Specifically, the silk fibroin / platelet-rich fibrin mixed aqueous solution was spin-coated at 5000 rpm for 60 s; the dispersion of MXene containing strontium and selenium-containing silk fibroin nanofibers was spin-coated at 3000 rpm for 20 s. Alternating spin-coating continued until the composite coating thickness reached 50 μm, with the outermost layer of the composite film consisting of silk fibroin, strontium, and selenium-containing MXene particles.

[0028] 4. Immerse the silicon wafer from step 3 in acetone for 2 hours, then peel the composite film off the silicon wafer. After drying, place the composite film at 40°C and 90% humidity for 36 hours. A conductive, bone-regenerating multilayer composite film is obtained. Comparative Example 1

[0029] The basic steps are the same as in Example 1, except that platelet-rich fibrin powder was not added. Comparative Example 2

[0030] The basic steps are the same as in Example 1, except that a dispersion of MXene containing strontium and selenium-containing silk fibroin nanofibers was not spin-coated. Comparative Example 3

[0031] The basic steps are the same as in Example 1, except that the dispersion of MXene containing strontium and selenium-containing silk fibroin nanofibers loaded on the surface is replaced with a dispersion of MXene particles (without any modification or loading). Comparative Example 4

[0032] The basic steps are the same as in Example 1, except that the dispersion of MXene containing strontium and selenium-containing silk fibroin nanofibers loaded on the surface is replaced with a dispersion of MXene containing strontium / selenium but without silk fibroin modification on the surface.

[0033]

[0034]

[0035]

[0036] Figure 1(a) shows the SEM image of MXene, and Figure 1(b) shows the SEM image of MXene (MXene@RSF) with strontium and selenium-containing silk fibroin nanofibers loaded on its surface. As can be seen from the figures, compared to MXene, the modified MXene has silk fibroin fibers on its surface, and these fibers overlap. Figure 1(c) shows the infrared images of MXene and MXene with strontium and selenium-containing silk fibroin nanofibers loaded on its surface, which also demonstrates that silk fibroin was successfully loaded onto the MXene surface. Figure 1(d) compares the electrical conductivity of MXene and MXene (MXene@RSF) with strontium and selenium-containing silk fibroin nanofibers loaded on its surface. It can be seen that compared to MXene, the electrical conductivity of the modified MXene is reduced, but it still retains conductivity.

[0037] Figure 2 shows the CCK-8 results of MC3T3-E1 osteoblasts co-cultured with samples from Examples 1-3 and Comparative Examples 1-4 for 3 days. As can be seen from the figure, cell proliferation gradually increased from Example 1 to Example 3. Comparing Example 1 and Comparative Example 1, cell proliferation decreased without the addition of platelet-rich fibrin. Comparing Example 1 and Comparative Example 2, cell proliferation also decreased without the addition of MXene with strontium and selenium-containing silk fibroin nanofibers on its surface. Comparing Example 1, Comparative Example 3, and Comparative Example 4, loading silk fibroin fibers onto the surface of MXene promotes cell proliferation.

[0038] Figure 3 shows the CCK-8 results of MC3T3-E1 osteoblasts and the sample from Example 1 after co-culturing for 3 days with and without 808nm near-infrared (NIR) laser irradiation for 5 min. It can be seen that the addition of NIR irradiation significantly promotes cell proliferation.

[0039] Figure 4 shows the growth morphology of MC3T3-E1 osteoblasts after co-culturing with different samples for 3 days (Figure 4(a) Comparative Example 2, Figure 4(b) Comparative Example 3, Figure 4(c) Comparative Example 4, Figure 4(d) Example 1, Figure 4(e) Example 1 (irradiated with 808nm laser for 5 min), Figure 4(f) Example 3). As shown in Figures 4(a) to 4(d), when the composite membrane lacks MXene with strontium and selenium-containing silk fibroin nanofibers on its surface, the cell number is the lowest and the cell spreading area is small; after coating with MXene (unmodified), the cell number and spreading area increase slightly; when the composite membrane has MXene with strontium and selenium-containing silk fibroin nanofibers on its surface, both the cell number and morphology improve. As shown in Figures 4(d) to 4(f), with the increase of MXene concentration with strontium and selenium-containing silk fibroin nanofibers on the surface of the composite membrane, the cell number increases and the spreading area increases. Furthermore, as shown in Figures 4(d) and 4(e), the number of cells and the spreading area both increased significantly after NIR irradiation.

[0040] Table 1 shows the sheet resistance of samples from Examples 1-3 and Comparative Examples 1-4. Data from Examples 1-3 indicate that as the concentration of MXene containing strontium and selenium-containing silk fibroin nanofibers on the surface increases, the surface resistance of the composite membrane decreases, and the conductivity increases. Examples 1 and Comparative Example 1 show that the content of platelet-rich fibrin has almost no effect on the conductivity of the composite membrane. Examples 1 and Comparative Example 2 show that the presence of MXene containing strontium and selenium-containing silk fibroin nanofibers on the surface is the main reason for the conductivity of the composite membrane. Examples 1, Comparative Examples 3 and 4 show that loading silk fibroin fibers onto the MXene surface slightly increases the resistance of the composite membrane, but loading strontium / selenium ions has no effect on the conductivity of the composite membrane.

[0041] Table 2 shows the hemolysis rates of samples from Examples 1-3 and Comparative Examples 1-4. As can be seen from the table, in Examples 1-3, the hemolysis rate of all membranes was less than 2%, indicating good blood compatibility. Furthermore, the hemolysis rate of the composite membrane decreased with increasing platelet-rich fibrin content. Examples 1 and Comparative Example 1 show that the absence of platelet-rich fibrin increases the hemolysis rate. Examples 1 and Comparative Example 2 show that without spin-coating a dispersion of MXene containing strontium and selenium-containing silk fibroin nanofibers, the hemolysis rate of the composite membrane was slightly lower than that of Example 1. Examples 1, Comparative Examples 3 and 4 show that loading silk fibroin fibers and strontium / selenium ions onto the MXene surface is beneficial for reducing the hemolysis rate of the material.

[0042] Table 3 shows the tensile properties of samples from Examples 1-3 and Comparative Examples 1-4. As can be seen from the table, in Examples 1-3, the tensile strength of the composite membrane gradually increases with increasing coating silk fibroin concentration, concentration of the MXene dispersion containing strontium and selenium-containing silk fibroin nanofibers, and coating thickness. Examples 1 and Comparative Example 1 show that the platelet-rich fibrin content has little effect on the tensile properties of the composite membrane. Examples 1 and Comparative Example 2 show that the presence of MXene containing strontium and selenium-containing silk fibroin nanofibers helps improve the tensile properties of the composite membrane. Examples 1, Comparative Examples 3 and 4 show that loading silk fibroin fibers onto the MXene surface helps improve the tensile properties of the composite membrane, but loading strontium / selenium ions has little effect on the mechanical properties.

[0043] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent transformations or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a conductive, bone-regenerating multilayer composite membrane, characterized in that, Includes the following steps: A silk fibroin aqueous solution was uniformly mixed with platelet-rich fibrin to obtain a silk fibroin / platelet-rich fibrin mixed aqueous solution; a dispersion of MXene containing strontium and selenium-containing silk fibroin nanofibers was prepared; the silk fibroin / platelet-rich fibrin mixed aqueous solution and the dispersion of MXene containing strontium and selenium-containing silk fibroin nanofibers were alternately spin-coated onto a silicon wafer with a cellulose acetate layer on the surface by alternating spin-coating technology, and repeated multiple times to form a composite film of a certain thickness; the silicon wafer obtained in step (3) was placed in acetone to dissolve and remove the cellulose acetate layer, and the composite film was peeled off from the silicon wafer. After drying, the composite film was treated with water vapor to obtain a conductive multilayer composite film that promotes bone regeneration.

2. The method for preparing a conductive, bone-regenerating multilayer composite membrane according to claim 1, characterized in that: In step (1), platelet-rich fibrin powder is added to a silk fibroin aqueous solution with a mass fraction of 2-6% in a certain proportion, and the mixture is stirred slightly to make the two evenly mixed to obtain a silk fibroin / platelet-rich fibrin mixed aqueous solution; wherein, the mass ratio of platelet-rich fibrin powder to silk fibroin is 0.01-5:

100.

3. The method for preparing a conductive, bone-regenerating multilayer composite membrane according to claim 1, characterized in that, In step (2), the specific preparation process of the MXene dispersion with strontium and selenium-containing silk fibroin nanofibers loaded on the surface is as follows: S1, Sodium selenite and strontium chloride are dissolved in a 4-8% (w / w) aqueous solution of silk fibroin; wherein the concentrations of sodium selenite and strontium chloride in the mixed solution are 0.01-5 mol / L and 0.01-5 mol / L, respectively. S2. After ultrasonically dispersing the monolayer MXene in water, add the mixture obtained in S1 to make the mass ratio of MXene to silk fibroin 0.01-1:10, and mix evenly; S3. Concentrate the mixture obtained in S2 at 60℃. When the concentration of silk fibroin in the mixture is 20-30%, take it out, add deionized water to dilute it, so that the mass fraction of silk fibroin in the mixture is reduced to 1-4%, and then treat it again at 60℃ for 24 hours; S4. Centrifuge the mixture obtained in S3, remove the supernatant, and obtain MXene with strontium and selenium-containing silk fibroin nanofibers on the surface. After freeze-drying, disperse it in methanol to obtain a dispersion of MXene with strontium and selenium-containing silk fibroin nanofibers on the surface with a concentration of 0.1-1%.

4. The method for preparing a conductive, bone-regenerating multilayer composite membrane according to claim 1, characterized in that: In step (3), the specific conditions for a single spin coating of the silk fibroin / platelet-rich fibrin mixed aqueous solution are: spin coating speed of 2000-5000 rpm and time of 10-60 s; the specific conditions for a single spin coating of the dispersion of MXene containing strontium and selenium silk fibroin nanofibers loaded on the surface are: spin coating speed of 1000-3000 rpm and time of 10-20 s; the thickness of the composite film is 1-50 μm, and the outermost layer of the composite film is MXene particles of silk fibroin, strontium and selenium; the thickness of the cellulose acetate layer is 100-500 nm.

5. The method for preparing a conductive, bone-regenerating multilayer composite membrane according to claim 1, characterized in that: In step (4), the silicon wafer is placed in acetone and the immersion time is 0.1-2 hours.

6. The method for preparing a conductive, bone-regenerating multilayer composite membrane according to claim 1, characterized in that: In step (4), the conditions for the water vapor treatment are: temperature of 30-40℃, humidity of 60-90%, and treatment time of 24-36 hours.

7. The application of a conductive, bone-regenerating multilayer composite membrane prepared by the method of any one of claims 1-6 in bone repair.

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