Degradable piezoelectric nanofiber membrane for bone repair as well as preparation method and application of degradable piezoelectric nanofiber membrane

Abstract

The invention relates to a degradable piezoelectric nanofiber membrane for bone repair and a preparation method and application thereof.The preparation method comprises the following steps that S1, a PLLA solution and MXene nanosheets are mixed, heated and stirred to obtain a uniform PLLA / MXene mixed solution, and the molecular weight of PLLA in the PLLA solution is 80000-120000; s2, carrying out electrostatic spinning on the PLLA / MXene mixed solution to obtain a PLLA / MXene nanofiber membrane semi-finished product; and S3, carrying out annealing, cooling, heat treatment and secondary cooling on the PLLA / MXene nanofiber membrane semi-finished product to obtain a PLLA / MXene nanofiber membrane finished product, namely the degradable piezoelectric nanofiber membrane. Compared with the prior art, the nanofiber membrane prepared by the method has good biocompatibility, degradability, piezoelectric property and mechanical property, can effectively promote bone tissue regeneration, and provides better material selection for bone injury repair.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and in particular to a degradable piezoelectric nanofiber membrane for bone repair, and a preparation method and application thereof. Background Art

[0002] Traditional methods for bone repair face numerous limitations. Autologous bone transplantation is limited by the number of donors, and the harvesting process can be traumatic for the patient. Allogeneic bone transplantation faces the risk of immune rejection and disease transmission. And synthetic bone substitutes have limited ability to induce bone regeneration. In recent years, nanofiber membranes have become a research hotspot due to their unique advantages. Their high surface area and porous structure facilitate cell adhesion and proliferation, providing a favorable microenvironment for bone tissue regeneration. Piezoelectric materials generate electrical signals when subjected to mechanical stress, which can promote cell proliferation, differentiation, and tissue regeneration. Incorporating piezoelectric properties into nanofiber membranes holds promise for the development of a novel bone repair material that can actively promote bone tissue regeneration and repair. However, existing materials suffer from poor degradability, unstable piezoelectric properties, and suboptimal cytocompatibility, limiting their clinical application in bone repair. Therefore, developing a biodegradable piezoelectric nanofiber membrane with superior performance for bone repair has significant clinical significance and application value.

[0003] MXene is a novel two-dimensional transition metal carbide or carbonitride with excellent electrical and thermal properties, making it widely used in sensors, energy storage, and other fields. Its abundant surface functional groups (such as -F, =O, and -OH) lend it excellent biocompatibility and promote cell-material interactions. Furthermore, MXene can generate a piezoelectric effect when subjected to external forces, potentially providing additional electrical stimulation for bone repair and accelerating bone regeneration.

[0004] Poly (L-lactic acid) (PLLA) is a commonly used biodegradable polymer material with excellent biocompatibility and biodegradability. Its degradation products are safe, non-toxic, and gradually metabolized and excreted in the body. Its excellent mechanical properties can provide some mechanical support for bone repair. However, the piezoelectric properties of a single PLLA nanofiber membrane are insufficient to fully meet the demand for material functional diversity during bone repair.

[0005] Combining PLLA and MXene to create nanofiber membranes has the potential to leverage the strengths of both materials, resulting in a bone repair material with excellent biocompatibility, biodegradability, piezoelectric properties, and sufficient mechanical strength. However, current research on this topic is incomplete, and material performance still needs to be improved. Therefore, developing a high-performance, biodegradable PLLA / MXene piezoelectric nanofiber membrane is of great significance.

[0006] Patent publication No. CN119015516A discloses a double-layer oral repair membrane material and a preparation method thereof, which comprises the following steps: 1) dispersing polylactic acid microspheres containing osteogenic active material in a methylacrylated protein aqueous solution to obtain a first mixture; spreading the first mixture, ultrasonic vibration, and drying to obtain a protein film; 2) ultrasonic treatment of nanoscale osteogenic active material powder and nanometer piezoelectric inorganic material in a surface modifier aqueous solution to obtain surface-modified nanometer powder; mixing the surface-modified nanometer powder, poly-L-lactic acid and a second solvent, and ultrasonic vibration to obtain an electrospinning solution; 3) forming a layer of fiber membrane on the protein film by electrospinning the electrospinning solution, then washing, freeze-drying to obtain a double-layer oral repair membrane material. However, the membrane material is a double-layer membrane structure, its biological activity depends on the loading of drugs or bioactive substances in the inner layer, and as an oral repair membrane material, it is often used for periodontal tissue defect, tooth extraction socket bone regeneration, and soft tissue barrier isolation, and there are problems of interlayer stability and drug controlled release.

[0007] Patent publication No. CN114748688A discloses a single-atom copper catalyst loaded artificial bone material and application, which is composed of left-handed poly-lactic acid powder, bioactive glass powder and SA-Cu-MXene powder. In the single-atom copper catalyst loaded artificial bone material, the mass percentage contents of the left-handed poly-lactic acid powder, bioactive glass powder and SA-Cu-MXene powder are 89.25-89.75%, 10.0% and 0.25-0.75%, respectively. However, in order to have anti-tumor performance, the artificial bone material adds copper ions, which has the adverse effect of inhibiting bone metabolism; the uniformity of the nanofiber topology is poor, the BMSCs adhesion density is low, and the SA-Cu-MXene copper-containing catalyst may cause toxicity and affect the normal growth of bone cells if it stays for a long time. SUMMARY

[0008] The purpose of the present application is to overcome the defects of the prior art and provide a degradable piezoelectric nanofiber membrane for bone repair and a preparation method and application thereof, so that the prepared nanofiber membrane has good biocompatibility, degradability, piezoelectric performance and mechanical properties, can effectively promote bone tissue regeneration, and provides a better material selection for bone injury repair.

[0009] The purpose of the present application can be achieved by the following technical solutions:

[0010] One of the technical solutions of the present application is to provide a preparation method of a degradable piezoelectric nanofiber membrane for bone repair, comprising the following steps:

[0011] S1, mixing PLLA solution and MXene nanosheet, heating and stirring to obtain a uniform PLLA / MXene mixed solution;

[0012] S2, obtaining a PLLA / MXene nanofiber membrane semi-product by electrospinning the PLLA / MXene mixed solution;

[0013] S3, obtaining a PLLA / MXene nanofiber membrane product, i.e., a degradable piezoelectric nanofiber membrane, by annealing, cooling, heat treatment and secondary cooling of the PLLA / MXene nanofiber membrane semi-product, and completing the process.

[0014] Further, in step S1, the mass ratio of PLLA to MXene nanosheet in the PLLA solution is 100:0.1-1.2.

[0015] Further, in step S1, the solute of the PLLA solution is PLLA, and the molecular weight is 80000-120000.

[0016] The solvent of the PLLA solution includes one or more of tetrahydrofuran and N,N-dimethylformamide.

[0017] Further, in step S1, the mass concentration of the PLLA solution is 8wt%.

[0018] Further, in step S1, the chemical formula of the MXene nanosheet is MXnT n+1 X n T x ;

[0019] wherein M represents a transition metal, including Ti, Mo, V, Nb; X represents C and / or N, and n is 1-3; T x represents a surface group, including -F, =O, -OH.

[0020] Further, the MXene nanosheet is Ti3C2T x .

[0021] Further, in step S1, the lateral size of the MXene nanosheet can be 0.3-1.2μm, the average size is 0.4-0.6μm, and the thickness is 0.8-1.5nm. This size range can ensure uniform dispersion of MXene in the nanofiber membrane and fully exert its piezoelectric performance and reinforcing effect.

[0022] Further, in step S1, the concentration of the PLLA / MXene mixed solution is 8.1wt%-8.9wt%, and in this concentration range, the viscosity and surface tension of the solution are suitable, which is conducive to the stable performance of the electrospinning process.

[0023] Furthermore, in step S2, the specific parameters of the electrospinning are: voltage 12-18 kV, receiving distance 12-18 cm, solution flow rate 0.8-1.5 mL / h.

[0024] During the electrospinning process, the PLLA molecular chains are stretched and oriented under the action of the electric field force, interweaving with the MXene nanosheets to form a nanofiber structure. The two-dimensional layered structure and rich functional groups of MXene enable it to form strong interactions with PLLA molecules, such as hydrogen bonds, which enhance the mechanical properties of the nanofiber membrane. At the same time, the piezoelectric properties of MXene cause the nanofiber membrane to produce a piezoelectric effect when subjected to external forces (such as pressure generated by human movement, stress from muscle contraction, etc.). The generated electrical signals can stimulate the proliferation, differentiation and migration of bone cells, promoting the repair and regeneration of bone tissue. The degradability of PLLA ensures that the nanofiber membrane is gradually absorbed by the human body after bone repair is completed, avoiding the trouble of secondary surgery for removal.

[0025] Furthermore, in step S3, the annealing temperature is 100-120° C., and the annealing time is 2-20 hours; the heat treatment temperature is 140-180° C., and the heat treatment time is 2-20 hours.

[0026] Furthermore, in step S3, the temperature after cooling is room temperature, and the temperature after secondary cooling is room temperature.

[0027] The second technical solution of the present invention is to provide a degradable piezoelectric nanofiber membrane for bone repair, characterized in that it is prepared by the preparation method.

[0028] Furthermore, the PLLA / MXene nanofiber membrane is formed by PLLA into a continuous nanofiber network, and MXene nanosheets are uniformly dispersed inside or on the surface of the nanofiber network in the form of intercalation or coating.

[0029] The third technical solution of the present invention is to provide a degradable piezoelectric nanofiber membrane for bone repair and its application in the field of bone repair.

[0030] Furthermore, the PLLA / MXene nanofiber membrane is used as a bone repair material for bone defect repair (such as skull regeneration) and complex wound healing promoted by ultrasonic stimulation.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] The preparation method of the application is simple, has wide application range, is safe in process, and the obtained product is good in uniformity, and is an efficient, fast and environment-friendly method for preparing nanofiber membranes. Moreover, the obtained degradable piezoelectric nanofiber membrane PLLA / MXene fiber membrane not only has high mechanical properties and electrical conductivity, but also generates a piezoelectric effect and releases an electric signal when subjected to external pressure or stress. These electric signals can stimulate the proliferation and differentiation of bone cells and promote the repair and regeneration of bone tissue.

[0033] (1) Good biocompatibility: PLLA itself has good biocompatibility, and the rich functional groups on the surface of MXene further promote cell adhesion and growth. Cell experiments show that osteoblasts can adhere, stretch and exhibit high proliferation activity on the nanofiber membrane, providing a favorable cell microenvironment for bone tissue regeneration.

[0034] (2) Efficient piezoelectricity promotes bone repair: MXene endows the nanofiber membrane with stable piezoelectric properties, and the electric signals generated when subjected to external force in a physiological environment can effectively promote the proliferation and differentiation of bone cells and accelerate the repair process of bone tissue. Animal experiments show that the amount of new bone formation in the experimental group using the nanofiber membrane of the application to repair bone damage is significantly higher than that in the control group, and the bone healing speed is faster. The piezoelectric effect of MXene is due to the charge separation of its two-dimensional layered structure under mechanical stress. Low concentration of MXene is dispersed and sparse, and cannot form a continuous charge path. While too high concentration of MXene is prone to aggregation, resulting in uneven local electric field distribution and attenuation of piezoelectric signal.

[0035] (3) Degradability and safety: The degradable nature of PLLA allows the nanofiber membrane to gradually degrade into harmless small molecular substances after bone repair is completed, which are metabolized and excreted by the body, avoiding inflammation and other problems that may be caused by long-term retention in the body, and improving the safety of the material. By low-temperature ultrasonic dispersion (add ice to prevent MXene from overheating) and DMF / THF mixed solvent, the oxidation of MXene is avoided, and the biological activity of the surface -F, -OH groups is ensured. BMSCs adhere tightly and stretch well on the membrane, and CCK8 test shows that the cell proliferation activity is not different from the blank group.

[0036] (4) Good mechanical properties: The surface functional groups (-OH, =O) of MXene form hydrogen bonds with the ester groups of PLLA, and the two-dimensional sheets of MXene are tightly combined with the molecular chains of PLLA through van der Waals force, which enhances the interface strength. The interaction between PLLA and MXene enhances the mechanical properties of the nanofiber membrane, which can provide certain mechanical support to the damaged site in the early stage of bone repair, helping to maintain the normal morphology and function of bone tissue and promoting bone healing.

[0037] (5) Preparation process is simple: the nanofiber membrane is prepared by using an electrospinning method, the method is simple to operate, low in cost, easy to realize large-scale production, and is conducive to the clinical transformation and popularization and application of the product.

[0038] (6) The BMSCs have high adhesion density: the PLLA / MXene nanofiber membrane of the application can construct a more uniform nanofiber topology, avoid the damage of BG particles to the fiber continuity, simulate the arrangement of the natural bone matrix fibers, and improve the adhesion density of BMSCs.

[0039] (7) MXene is significantly superior to traditional ceramic materials in biological activity and interface bonding strength, and at the same time avoids the mechanical defects caused by the brittleness of ceramic. The monolayer film realizes the functions of mechanical support, piezoelectric effect and degradation through the molecular-level compounding of PLLA and MXene, avoids the delamination risk (such as interlayer peeling caused by saliva erosion in the oral environment), at the same time, the electrospinning one-step forming is easy to control the parameters, and is suitable for large-scale production. The conductive network of MXene enhances the electrical signal transmission, directly stimulates bone cells, and the oral repair may pay more attention to the surface antibacterial or short-term stimulation, and the demand for electrical signals is lower.

[0040] (8) The PLLA prepared by electrospinning or melt forming usually has low crystallinity (even amorphous), resulting in poor mechanical properties (such as toughness, modulus) and heat resistance. Annealing can promote the rearrangement of molecular chains, form a more ordered crystal structure, and significantly improve the crystallinity. The performance of the alpha crystal form (high ordered structure) and the alpha' crystal form (loose disorder) of PLLA is significantly different. The annealing temperature and time can control the crystal form transformation: high-temperature annealing (such as above 120 DEG C) is more likely to form alpha crystal form, improve the modulus and heat resistance of the material. The piezoelectric response of the high-crystallinity PLLA nanofiber is enhanced after annealing, which is suitable for energy harvesting devices (such as nanogenerators), and the improvement of the piezoelectric coefficient d33 is closely related to the ordered arrangement of the crystalline phase. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The flow chart of the preparation method of the nanofiber membrane of the application;

[0042] Figure 2For the different nanofiber membrane of the real figure and the morphology characterization figure, (A) Example 1~5, comparative example 1, 2 The schematic diagram of the solution prepared, (B) The macroscopic graph of the piezoelectric nanofiber membrane prepared by comparative example 1 and the fiber microstructure under electron microscope scanning, (C) The macroscopic graph of the piezoelectric nanofiber membrane prepared by example 2 and the fiber microstructure under electron microscope scanning, (D) The macroscopic graph of the piezoelectric nanofiber membrane prepared by example 3 and the fiber microstructure under electron microscope scanning, (E) The macroscopic graph of the piezoelectric nanofiber membrane prepared by example 4 and the fiber microstructure under electron microscope scanning, (F) The macroscopic graph of the piezoelectric nanofiber membrane of example 5 and the fiber microstructure under electron microscope scanning, (G) The macroscopic graph of the piezoelectric nanofiber membrane of example 1 and the fiber microstructure under electron microscope scanning;

[0043] Figure 3 For the piezoelectric property test graph of nanofiber membrane, (A) The output voltage waveform of piezoelectric device based on example 1~5 and comparative example 1, 2 under US activation, (B) The peak-to-peak output voltage of the sensor made by the piezoelectric device based on example 1 under US activation (n=4 independent samples), (C) The output voltage waveform of the piezoelectric device based on example 1 (1.0wt% MXene content) activated under different ultrasonic intensity stimulation, (D) The peak-to-peak output voltage of the piezoelectric device based on example 1 (1.0wt% MXene content) activated under different ultrasonic intensity stimulation;

[0044] Figure 4 For testing the activity and proliferation of BMSCs cells on the nanofiber membrane of example 1 and comparative example 1, (A) The live and dead staining graph of BMSCs on different nanofiber membranes, (B) CCK8 experiment verifies the influence of different nanofiber membranes on cell activity, wherein Con: blank group, PLLA: comparative example 1, Ph-TM NFs: example 1, Con(+): blank group+ultrasonic stimulation, PLLA(+): comparative example 1+ultrasonic stimulation, Ph-TM NFs(+): example 1+ultrasonic stimulation;

[0045] Figure 5 For testing the migration and adhesion performance of BMSCs on different nanofiber membranes, (A) Transwell experiment, (B) SEM photograph of cell adhesion on nanofiber membrane under different conditions;

[0046] Figure 6 For the degradation effect graph of the nanofiber membrane of example 1 at different times, (A) 37℃, pH 7.4 PBS, (B) 60℃ pH 7.4 PBS;

[0047] Figure 7 For the tensile strength and elastic modulus graph of different nanofiber membranes;

[0048] Figure 8For the osteogenic differentiation potential of BMSCs in the examples, Comparative Example 1, blank group, (A) intracellular calcium levels of BMSCs with and without ultrasound stimulation for 20 minutes were detected by Fluo-4 staining, scale bar: 200 μm; (B) ALP staining of BMSCs cultured with or without ultrasound for 7 days, scale bar: 200 μm; (C) alizarin red staining of BMSCs at 21 days, scale bar: 200 μm; (D) quantitative evaluation of the average fluorescence intensity of Fluo-4 in each group; (E) quantitative evaluation of ALP positive area at 7 days; (F) quantitative evaluation of calcium nodules in alizarin red staining at 21 days; (G) Western blot analysis of core protein expression involved in osteogenesis; (H)-(I) Rt-qPCR analysis of classic osteogenic markers (OPN, RUNX2);

[0049] Figure 9 For the therapeutic effect of Ph-NFs TM in the rat calvarial defect model, (A) schematic diagram of rat treatment scheme; (B) voltage of Ph-TM NFs generated by ultrasound stimulation before and after implantation; (C) 3D reconstruction micro-CT scan image of rat mandibular defect; (D) quantitative micro-CT analysis of BV / TV; (E) quantitative micro-CT analysis of Tb.N (n=3), data are expressed as SD±mean, one-way ANOVA was used for statistical analysis, ***p<0.00, *p<0.05, n=3;

[0050] Figure 10 For (A) H&E staining images of mandibular defects at 6 and 12 weeks after surgery, low and high magnification; (B) Masson trichrome staining images of mandibular defects at 6 and 12 weeks after surgery; the letters B in the figure represent old bone tissue, N represents newly formed bone tissue, M represents nanofiber membrane, and F represents fibrous connective tissue. The scale bar is 500 μm (top), 100 μm (bottom);

[0051] Figure 11 For the d33 piezoelectric property test of PLLA nanofiber membrane (Comparative Example 1) before and after annealing. DETAILED DESCRIPTION

[0052] The application will be described in detail below with reference to the accompanying drawings and specific examples. The present embodiment is implemented on the premise of the technical scheme of the present application, and gives a detailed implementation and specific operation process, but the protection scope of the present application is not limited to the following examples. Based on the given examples, all other examples obtained by those of ordinary skill in the art without making creative efforts are within the scope of the present application.

[0053] Unless otherwise indicated, the reagents, methods, instruments and apparatus employed in the present application are those conventional in the art.

[0054] A preparation method of a degradable piezoelectric nanofiber membrane for bone repair, comprising the following steps:

[0055] S1, mixing PLLA solution and MXene nanosheet, heating and stirring to obtain uniform PLLA / MXene mixed solution;

[0056] S2, the PLLA / MXene mixed solution is obtained by electrospinning to obtain PLLA / MXene nanofiber membrane semi-finished product;

[0057] S3, the PLLA / MXene nanofiber membrane semi-finished product is annealed, cooled, heat treated and secondarily cooled to obtain PLLA / MXene nanofiber membrane finished product, i.e. degradable piezoelectric nanofiber membrane, which is completed.

[0058] Further, in step S1, the mass ratio of PLLA to MXene nanosheet in the PLLA solution is 100:0.1-1.2.

[0059] Further, in step S1, the solute of the PLLA solution is PLLA, and the molecular weight is 80000-120000.

[0060] The solvent of the PLLA solution includes one or more of tetrahydrofuran, N,N-dimethylformamide.

[0061] In some specific embodiments, in step S1, the mass concentration of the PLLA solution is 8wt%.

[0062] In some specific embodiments, in step S1, the chemical formula of the MXene nanosheet is M n+1 X n T x ;

[0063] Wherein M represents transition group metal, including Ti, Mo, V, Nb; X represents C and / or N, n is 1-3; T x represents surface group, including -F, =O, -OH.

[0064] In some specific embodiments, the MXene nanosheet adopts Ti3C2T x .

[0065] In some specific embodiments, in step S1, the lateral size of the MXene nanosheet can be 0.3-1.2 μm, the average size can be 0.4-0.6 μm, and the thickness can be 0.8-1.5 nm. This size range can ensure the uniform dispersion of MXene in the nanofiber membrane and fully exert its piezoelectric performance and reinforcing effect.

[0066] In some specific embodiments, in step S1, the concentration of the PLLA / MXene mixed solution can be 8.1wt%-8.9wt%. Within this concentration range, the viscosity and surface tension of the solution are suitable, which is conducive to the stable performance of the electrospinning process.

[0067] In some specific embodiments, in step S2, the specific parameters of the electrospinning are as follows: the voltage is 12-18 kV, the receiving distance is 12-18 cm, and the solution flow rate is 0.8-1.5 mL / h.

[0068] In the electrospinning process, the PLLA molecular chain is stretched and oriented under the action of the electric field force, and interweaves with the MXene nanosheet to form a nanofiber structure. The two-dimensional sheet structure and abundant functional groups of MXene enable it to form strong interactions with the PLLA molecules, such as hydrogen bonds, thereby enhancing the mechanical properties of the nanofiber membrane. At the same time, the piezoelectric properties of MXene enable the nanofiber membrane to produce a piezoelectric effect when subjected to external forces (such as pressure generated by human movement, stress caused by muscle contraction, etc.), and the generated electric signal can stimulate the proliferation, differentiation, and migration of bone cells, thereby promoting the repair and regeneration of bone tissue. The biodegradability of PLLA ensures that the nanofiber membrane is gradually absorbed by the human body after the bone repair is completed, thereby avoiding the trouble of secondary surgery for removal.

[0069] In some specific embodiments, in step S3, the annealing temperature is 100-120℃, and the annealing time is 2-20 h; the heat treatment temperature is 140-180℃, and the heat treatment time is 2-20 h.

[0070] In some specific embodiments, in step S3, the temperature after cooling is room temperature, and the temperature after secondary cooling is room temperature.

[0071] A biodegradable piezoelectric nanofiber membrane for bone repair, which is prepared by the preparation method.

[0072] In some specific embodiments, the PLLA / MXene nanofiber membrane is formed by a continuous nanofiber network of PLLA, and the MXene nanosheet is uniformly dispersed in the interior or surface of the nanofiber network in the form of intercalation or coating.

[0073] The application of a biodegradable piezoelectric nanofiber membrane for bone repair in the field of bone repair.

[0074] In some specific embodiments, the PLLA / MXene nanofiber membrane is used as a bone repair material for bone defect repair (such as skull regeneration), and is combined with ultrasonic stimulation to promote healing of complex wounds.

[0075] Each of the above embodiments can be implemented alone or in any two or more combinations.

[0076] The following describes specific embodiments.

[0077] Embodiment 1

[0078] A preparation method of a degradable piezoelectric nanofiber membrane for bone repair, comprising the following steps, as shown in Figure 1

[0079] S1, PLLA with a molecular weight of 120000 (Hangzhou Jetnofly Biotechnology Co., Ltd.; BP02) is dissolved in a mixed solvent of tetrahydrofuran and N, N-dimethylformamide (mass ratio 1:1) to prepare an 8wt% PLLA solution. MXene nanosheets are obtained by etching method. First, 2g of lithium fluoride powder (Sigma-Aldrich; CAS: 7789-24-4) is dissolved in 20ml of 9mmol / L hydrochloric acid solution, then 1g of Ti3AlC2 powder (Jilin Yi Yi Technology Co., Ltd.; CAS: 196506-01-01) is added, stirred at 50°C for 30 hours, the obtained solution is taken out, washed with water for 5 times, then ultrasonic treated for 1h, and the nanosheets are taken after centrifugation. The obtained Ti3C2T x MXene nanosheets (lateral size 0.3-1.2μm, average size 0.4-0.6μm, thickness 0.8-1.5nm) are added to the PLLA solution to make the concentration of MXene nanosheets 1wt%, ice is added in the ultrasonic instrument to prevent MXene from overheating and caking, after ultrasonic treatment for 30min, 60°C water bath heating and stirring for 1h, a mixed solution is obtained, the mixed solution is ultrasonic treated for 5min to remove bubbles, and a uniform PLLA / MXene mixed solution is obtained.

[0080] S2, the PLLA / MXene mixed solution is loaded into a syringe with a needle, and the electrospinning parameters are set as follows: voltage 14kV, receiving distance 15cm, solution flow rate 1mL / h, electrospinning is carried out, and the PLLA / MXene nanofiber membrane semi-finished product is collected on the receiving device.

[0081] S3, the PLLA / MXene nanofiber membrane semi-finished product is annealed at 105°C for 10h, cooled to room temperature, then heat treated at 160°C for 10h and cooled to room temperature, and the PLLA / MXene nanofiber membrane finished product, i.e. the degradable piezoelectric nanofiber membrane, is obtained.​

[0082] Example 2

[0083] Compared with Example 1, most of the steps are the same, except that the concentration of MXene nanosheets in step S1 is adjusted to 0.2 wt %.

[0084] Example 3

[0085] Compared with Example 1, most of the steps are the same, except that the concentration of MXene nanosheets in step S1 is adjusted to 0.4 wt %.

[0086] Example 4

[0087] Compared with Example 1, most of the steps are the same, except that the concentration of MXene nanosheets in step S1 is adjusted to 0.6 wt %.

[0088] Example 5

[0089] Compared with Example 1, most of the steps are the same, except that the concentration of MXene nanosheets in step S1 is adjusted to 0.8 wt %.

[0090] Comparative Example 1

[0091] A method for preparing a PLLA nanofiber membrane, compared with Example 1, without adding MXene nanosheets, specifically comprising the following steps:

[0092] S1. Dissolve PLLA (molecular weight 120,000) in a 1:1 mixed solvent of tetrahydrofuran and N,N-dimethylformamide to prepare an 8 wt% PLLA solution. Ultrasonicate for 30 minutes, then heat in a 60°C water bath with stirring for 1 hour, and ultrasonicate for 5 minutes to remove bubbles to obtain a uniform PLLA solution.

[0093] S2. The PLLA solution was loaded into a syringe with a needle, and the electrospinning parameters were set as follows: voltage of 14 kV, receiving distance of 15 cm, and solution flow rate of 1 mL / h. Electrospinning was performed and the semi-finished PLLA nanofiber membrane was collected on the receiving device.

[0094] S3. Anneal the PLLA nanofiber membrane semi-finished product at 105° C. for 10 h, cool it to room temperature, then heat-treat it at 160° C. for 10 h and cool it to room temperature to obtain a PLLA nanofiber membrane finished product.

[0095] Comparative Example 2

[0096] Compared with Example 1, most of the steps are the same, except that the concentration of MXene nanosheets in step S1 is adjusted to 1.2 wt %.

[0097] Comparative Example 3

[0098] Most of them are the same as compared with Example 1, except that the PLLA / MXene nanofiber membrane semi-finished product is not annealed in step S3.

[0099] Performance test:

[0100] The nanofiber membranes prepared in the examples and comparative examples are subjected to morphology characterization, piezoelectric performance test, cell compatibility test, migration and adhesion performance test, degradation performance test, tensile strain mechanical property test, bone repair performance test, and annealing effect test.

[0101] I. Morphology characterization

[0102] The morphology of the nanofiber membranes obtained in Examples 1-5 and Comparative Examples 1 and 2 is characterized, Figure 2 (A) Schematic diagram of the solution prepared in Examples 1-5, Comparative Examples 1 and 2, Figure 2 (B) Macroscopic view of the piezoelectric nanofiber membrane prepared in Comparative Example 1 and fiber microstructure under electron microscope scanning, Figure 2 (C) Macroscopic view of the piezoelectric nanofiber membrane prepared in Example 2 and fiber microstructure under electron microscope scanning, Figure 2 (D) Macroscopic view of the piezoelectric nanofiber membrane prepared in Example 3 and fiber microstructure under electron microscope scanning, Figure 2 (E) Macroscopic view of the piezoelectric nanofiber membrane prepared in Example 4 and fiber microstructure under electron microscope scanning, Figure 2 (F) Macroscopic view of the piezoelectric nanofiber membrane prepared in Example 5 and fiber microstructure under electron microscope scanning, Figure 2 (G) Macroscopic view of the piezoelectric nanofiber membrane prepared in Example 1 and fiber microstructure under electron microscope scanning, Figure 2 (H) Macroscopic view of the piezoelectric nanofiber membrane prepared in Comparative Example 2 and fiber microstructure under electron microscope scanning. Figure 3 It can be seen that the number of MXene nanosheets on the fibers increases with the increasing concentration of MXene.

[0103] II. Piezoelectric performance test

[0104] The piezoelectric performance of the nanofiber membranes obtained in Examples 1-5 and Comparative Examples 1 and 2 is tested. The piezoelectric test system is used to apply ultrasonic stimulation to the nanofiber membrane, and the voltage signal generated is measured. The degradable piezoelectric nanofiber membrane prepared in Example 1 is cut into 2x2cm 2 pieces. In order to measure the piezoelectric properties, these pieces are clamped between two copper foil electrodes. Then the copper wire is connected to the copper foil electrode through the conductive silver glue, and finally the whole device is packaged with a polyimide film.

[0105] Figure 3 Piezoelectric property test diagram for nanofiber membrane, Figure 3(A) Output voltage waveform of piezoelectric devices based on Examples 1-5 and Comparative Examples 1, 2 under US activation, Figure 3 (B) Peak-to-peak output voltage of sensors made from piezoelectric devices based on Example 1 under US activation (n = 4 independent samples), Figure 3 (C) Output voltage waveform of piezoelectric devices based on Example 1 (1.0 wt% MXene content) activated under stimuli with different ultrasonic intensities, Figure 4 (D) Peak-to-peak output voltage of piezoelectric devices based on Example 1 (1.0 wt% MXene content) activated under stimuli with different ultrasonic intensities. The results show that the voltage signals generated by the nanofiber membranes obtained from Examples 1-5 and Comparative Examples 1, 2 were ~500 mV (peak-to-peak), ~200 mV, ~260 mV, ~320 mV, ~400 mV, ~160 mV, ~320 mV, respectively, when subjected to ultrasonic stimuli at 1 MHz. The voltage signals generated by Example 1 were ~300 mV, ~500 mV, ~680 mV, respectively, under stimuli with different ultrasonic intensities.

[0106] III. Cell compatibility test

[0107] Cell compatibility tests were performed on the nanofiber membranes obtained from Example 1 and Comparative Example 1. After sterilization and vacuum storage of the nanofiber membranes, BMSCs were seeded on the nanofiber membranes, and live-dead cell staining and CCK8 experiments were performed. The absorbance values were measured using a microplate reader after 1 day, 3 days, and 5 days of culture, respectively, and BMSCs without nanofiber membranes were used as a blank control (con). The BMSCs were obtained from the bone marrow cavity of SD rats. Specifically, two-week-old male (Sprague-Dawley) SD rats were sacrificed by cervical dislocation, and then their whole bodies were immersed in 75% alcohol for 5 minutes for disinfection. Then the hind legs were removed, and the femur and tibia were taken out. After stripping the surrounding soft tissue, the epiphysis was excised, and the bone marrow cavity was flushed three times with complete a-MEM medium containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin / streptomycin (Gibco, USA) to obtain fresh bone marrow. The mixture was centrifuged at 3000 rpm for 10 minutes, and the supernatant was discarded. The cells were suspended in 1 milliliter of complete medium and inoculated in a 100 millimeter culture dish, and cultured in a humidified environment at 37°C and 5% carbon dioxide. The culture medium was replaced after four days of culture to promote the formation of initial BMSCs colonies and remove non-adherent irrelevant cells. To support osteogenic differentiation, osteogenic complete medium containing ascorbic acid (50 μg ml-1; Sigma-Aldrich, USA), 10 mM β-glycerophosphate (Sigma-Aldrich, USA), and 10 nM dexamethasone (Sigma-Aldrich, USA) was used, and the medium was changed every two days.

[0108] Figure 4 (A) is a live and dead staining diagram of BMSCs on different fiber membranes. Specifically, 1x105 BMSCs were inoculated into a 24-well plate for 6 hours for complete adhesion, and then the membranes of each group were covered, which had been sterilized by ultraviolet irradiation. After 48 hours of culture, the cell survival on the membranes was evaluated by using a calcein / PI cell viability / cytotoxicity assay kit (Beyotime, Shanghai, China) through a Leica Thunder Imager (Leica Microsystems, Wetzlar, Germany).

[0109] Figure 4 (B) is a result diagram of CCK8 experiment verifying the influence of different fiber membranes on the activity of BMSCs cells. For Figure 4 The ultrasonic treatment in (B) is in the form of low-frequency ultrasound, and the ultrasonic intensity is 1 MHz. On the one hand, the nanofiber membrane generates piezoelectricity, and on the other hand, many related studies have proved the auxiliary effect of low-frequency ultrasound on bone tissue healing.

[0110] The results show that the cell survival rate on the nanofiber membrane prepared by example 1 and comparative example 1 has no obvious difference with the blank group ( Figure 4 A), which indicates that the PLLA / MXene nanofiber membrane of the present application will not affect the proliferation and activity of cells, and at the same time, the piezoelectricity generated under ultrasonic stimulation plays a certain role in promoting proliferation. Figure 5 B)

[0111] Four, migration and adhesion performance test

[0112] Figure 5 The migration and adhesion performance of BMSCs on different fiber membranes (example 1, comparative example 1) were tested. Figure 5 (A) is a Transwell experiment, Figure 6 (B) is a SEM photograph of cells adhering to the fiber membrane under different conditions. It shows that cells can adhere to nanofibers, and in the Transwell experiment, it can be seen that the number of cells migrating in the example 1 (+ultrasound) group is the largest.

[0113] Five, degradation performance test

[0114] One key advantage of the nanofiber membrane is its programmable biodegradation profile in physiological media. The nanofiber membrane obtained from Example 1 was subjected to degradation performance test: the nanofiber membrane was placed in PBS solution (phosphate buffered saline, physiological pH range (7.2-7.6), used to simulate the degradation process of the nanofiber membrane in vivo) and incubated in a constant temperature shaking incubator at 37°C. Accelerated degradation tests (A, 37°C, pH 7.4 PBS; B, 60°C, pH 7.4 PBS) showed the structure progressive disintegration, partial fragmentation was observed at 28 days, and almost complete matrix decomposition was observed at 91 days Figure 7 B).

[0115] VI. Mechanical property test

[0116] The Ti3C2T x not only as a local polarizer to enhance the piezoelectric response, but also as a bridging agent to improve the flexibility and mechanical integrity of the piezoelectric film, the nanofiber membrane was subjected to mechanical property test, Figure 8 The stress-strain curve shown in the figure indicates that the tensile strength of the piezoelectric film is positively correlated with the content of Ti3C2T x The higher the content of Ti3C2T x The higher the content of Ti3C2T x mediated by interfacial hydrogen bonds and limited polymer chain mobility.

[0117] VII. Bone repair performance test

[0118] The nanofiber membranes obtained from Example 1 and Comparative Example 1 were subjected to bone repair performance test. In vitro, by co-culturing BMSCs with the nanofiber membrane, the intracellular calcium ions were fluorescently stained using calcium ion fluorescent probe Fluo-4AM (S1061S, Biotek, China). With the stimulation of ultrasonic instrument, the power was 0.3 MHz, and the calcium ion influx was a timely response. After the cells were cultured to an appropriate density in the culture plate, they were treated with ultrasound for 20 minutes, then the culture medium was removed from the well, washed with PBS for 3 times, and 100 μL of Fluo-4 calcium reagent was added. Then the cells were incubated at 37°C for 30 minutes, observed under a fluorescence microscope, and the average fluorescence intensity was analyzed using Image J. After ultrasonic stimulation, the fluorescence intensity of BMSCs on the nanofiber membrane was observed.

[0119] The intracellular calcium level of BMSCs with and without ultrasonic stimulation (+US, -US) for 20 minutes was detected by Fluo-4 staining, scale bar = 200 μm, as Figure 8 (A).

[0120] ALP staining of BMSCs cultured with or without ultrasound on day 7 (BCIP / NBT alkaline phosphatase colorimetric kit, Beyotime, China). Scale bar, 200 μm, as shown in Figure 8 (B).

[0121] Alizarin red staining of BMSCs on day 21 (Alizarin red S staining solution (0.2%, pH 8.3), Biyuntian, China). Scale bar, 200 μm. Figure 8 (C).

[0122] Quantitative evaluation of Fluo-4 mean fluorescence intensity (MFI) in each group, data are expressed as SD ± mean, one-way ANOVA with Tukey's multiple comparison test, ***p < 0.00, *p < 0.05, n = 3, as shown in Figure 8 (D).

[0123] Quantitative evaluation of ALP-positive areas on day 7. Data are expressed as SD ± mean. One-way ANOVA with Turkey's multiple comparison test, **P < 0.01, *P < 0.05, n = 3. Figure 8 (E).

[0124] Quantitative evaluation of calcium nodules by Alizarin red staining on day 21. Data are expressed as mean ± SD. Two-way ANOVA with Turkey's multiple comparison test, ***P < 0.001 and *P < 0.05, n = 3. Figure 8 (F).

[0125] Western blot analysis of the expression of core proteins involved in osteogenesis Figure 8 (G) and RNA expression Figure 8 (H).

[0126] RT-qPCR analysis of upregulation of classic osteogenic markers (OPN, Runx2), n = 3 biologically independent samples; mean ± SD. *P < 0.05, one-way ANOVA, see Figure 8 (I).

[0127] Compared with other groups, the cells on the nanofiber membrane of Example 1 showed the strongest fluorescence intensity, while the fluorescence intensity of the blank group (no fiber membrane, no ultrasonic stimulation) did not change significantly, see Figure 8 (A), (D). The osteogenic effect was evaluated by ALP and ARS staining. ALP activity is generally known as an early osteogenic biomarker. After 7 days of incubation on the membrane, Comparative Example 1 and Example 1 showed a darker purple color, with Example 1 showing the highest level among all groups. Figure 9(B), (E). According to reports, calcium salt deposition and bone matrix mineralization are important stages of bone formation. The formation of calcium nodules in the extracellular matrix was analyzed by alizarin red S staining. After 21 days of culture, the calcium nodules of Example 1 were significantly improved compared with the blank group and Comparative Example 1, see Figure 9 (C), (F). To quantify the expression of each gene and protein in the cells, the expression of osteogenesis-related genes (OPN, Runx2) was quantified using Western blot and qPCR at day 7 Figure 9 (G)-(I)), where the piezoelectric effect produced by the combination of Example 1 and ultrasound stimulation significantly increased the expression of these genes and proteins. All these results indicate that the piezoelectric nanofiber membrane can promote the osteogenic differentiation of BMSCs under ultrasound.

[0128] Example 1 was tested in vivo by a rat calvarial defect model, where the nanofiber membrane of Example 1 was attached to the defect. Specifically, a circular bone defect with a diameter of 5 mm was created in the rat calvarium, and the piezoelectric biomembrane was implanted into the defect site by surgery. The experimental group (+US) received ultrasound stimulation (1 MHz, 0.3 W cm -2 , 20 min) for 4 days a week, as Figure 10 (A). The blank group (-US) was a simple bone defect model without intervention. At the same time, there was no difference in the voltage generated by the biomembrane before and after the application of ultrasound, indicating that a stable piezoelectric effect can still be generated after implantation Figure 10 (B)). Micro-CT scanning Figure 9 (C)), HE Figure 11 (A)) and Masson ​ (B)) staining were performed at 6 and 12 weeks to evaluate the amount of new bone and the healing of the defect, among other things. In addition, in the quantitative analysis, Example 1 with ultrasound stimulation showed enhanced bone regeneration, with the highest bone tissue volume / total tissue volume (BV / TV) value and trabecular number (Tb.N). At 12 weeks, the BV / TV and Tb.N levels of the Example 1 with ultrasound stimulation group were 77.017% and 1.5 mm, respectively, while those of the blank group were 51.87% and 1.42 mm, respectively, and those of the blank group were only 35.94% and 13.63 ± 0.92 mm, respectively. See ​ (D), (E). Compared with only making a defect without any additional treatment, the nanofiber membrane prepared in Example 1 showed more new bone and fiber formation at all time points when combined with ultrasound stimulation.

[0129] VIII. Annealing influence test

[0130] ​To test the d33 (piezoelectric performance) of the nanofiber membranes before and after annealing (Example 1 and Comparative Example 3), the membranes were cut into 2x2 squares and measured using a piezoelectric tester (ZJ-6AN Quasi-static d33 / d31 Meter). High-temperature annealing increases the crystallinity of PLLA, thereby improving its piezoelectric properties.

[0131] Although the present invention has been described in detail above using general explanations, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for preparing a degradable piezoelectric nanofiber membrane for bone repair, characterized in that: The following steps are involved: S1, mixing the PLLA solution and MXene nanosheets, heating and stirring to obtain a uniform PLLA / MXene mixed solution; S2, electrospinning the PLLA / MXene mixed solution to obtain a PLLA / MXene nanofiber membrane semi-finished product; S3. Annealing, cooling, heat treating, and secondary cooling the PLLA / MXene nanofiber membrane semi-finished product to obtain a PLLA / MXene nanofiber membrane finished product, i.e., a degradable piezoelectric nanofiber membrane.

2. The method for preparing a degradable piezoelectric nanofiber membrane for bone repair according to claim 1, characterized in that: In step S1, the mass ratio of PLLA to MXene nanosheets in the PLLA solution is 100:0.2-1.

2.

3. The method for preparing a degradable piezoelectric nanofiber membrane for bone repair according to claim 1, characterized in that: In step S1, the solute of the PLLA solution is PLLA with a molecular weight of 80,000 to 120,000; The solvent of the PLLA solution includes one or more of tetrahydrofuran and N,N-dimethylformamide, and the mass concentration of the PLLA solution is 2wt% to 20wt%.

4. The method for preparing a degradable piezoelectric nanofiber membrane for bone repair according to claim 1, characterized in that: In step S1, the chemical formula of the MXene nanosheet is M n+1 X n T x ; Wherein M represents a transition metal, including Ti, Mo, V, Nb; X represents C and / or N, and n is 1 to 3; T x Represents surface groups, including -F, =O, and -OH.

5. The method for preparing a degradable piezoelectric nanofiber membrane for bone repair according to claim 1, characterized in that: In step S1 , the MXene nanosheets may have a lateral size of 0.3 to 1.2 μm, an average size of 0.4 to 0.6 μm, and a thickness of 0.8 to 1.5 nm.

6. The method for preparing a degradable piezoelectric nanofiber membrane for bone repair according to claim 1, characterized in that: In step S2, the specific parameters of the electrospinning are: voltage 12-18 kV, receiving distance 12-18 cm, solution flow rate 0.8-1.5 mL / h.

7. The method for preparing a degradable piezoelectric nanofiber membrane for bone repair according to claim 1, characterized in that: In step S3, the annealing temperature is 100-120°C, and the heat treatment temperature is 140-180°C.

8. A degradable piezoelectric nanofiber membrane for bone repair, characterized in that: The compound is prepared by the preparation method according to any one of claims 1 to 7.

9. The degradable piezoelectric nanofiber membrane for bone repair according to claim 8, characterized in that: The PLLA / MXene nanofiber membrane product is formed by PLLA into a continuous nanofiber network, and MXene nanosheets are uniformly dispersed in the interior or surface of the nanofiber network in the form of intercalation or coating.

10. Use of the degradable piezoelectric nanofiber membrane for bone repair according to claim 8 in the field of bone repair.

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