A method for preparing piezoelectric nanofiber aerogel

Abstract

The application provides a preparation method of piezoelectric nanofiber aerogel, comprising the following steps: S1, zinc oxide is added into hexafluoroisopropanol, polyhydroxybutyrate is added after ultrasonic dispersion, and magnetic stirring is conducted to obtain a PHB@ZnO solution; S2, the PHB@ZnO solution is prepared into a nanofiber membrane through electrospinning, the nanofiber membrane is placed into a vacuum drying box for vacuum drying to obtain a PHB@ZnO nanofiber membrane; S3, the PHB@ZnO nanofiber membrane is added into deionized water, and a short PHB@ZnO nanofiber dispersion liquid is obtained after homogenization; S4, chitosan powder is added into deionized water, glacial acetic acid is added after stirring, and chitosan solution is obtained after continuous stirring; S5, the short PHB@ZnO nanofiber dispersion liquid, the chitosan solution and glutaraldehyde are uniformly mixed to obtain a mixture, the mixture is poured into a mold, the mold is placed into a freeze dryer for freeze drying to obtain piezoelectric nanofiber aerogel. The piezoelectric nanofiber aerogel prepared by the piezoelectric nanofiber aerogel has a porous structure and good piezoelectric performance, and can accelerate bone defect repair.

Description

Technical Field

[0001] This invention relates to an aerogel, and more particularly to a method for preparing a piezoelectric nanofiber aerogel. Background Technology

[0002] Bones are vital organs in the human body and are easily damaged. Open or comminuted fractures with large bone defects, nonunion fractures, and bone loss after osteomyelitis resection can all lead to bone defects, making bone defect repair a major clinical challenge. Human bone tissue is a natural piezoelectric material. Studies have shown that when bone tissue is under stress, the negatively charged side exhibits accelerated osteoblast proliferation and differentiation, indicating a bone repair state, while the positively charged side shows enhanced osteoclast activity, indicating a bone resorption state. Therefore, the piezoelectric effect of bone plays a crucial role in bone repair.

[0003] Currently, piezoelectric materials have great potential as novel bone substitutes. Piezoelectric polymers are mostly flexible, easy to process, and biocompatible, but their piezoelectric properties are relatively low, and their mechanical strength is often insufficient, making it difficult to reach the electrical stimulation threshold for promoting bone repair and to withstand physiological loads as bone implants. Therefore, it is currently recommended to combine polymers with high-strength inorganic ceramics to form piezoelectric composite materials, which can combine the advantages of both and serve as an important alternative material for artificial bone repair of bone defects.

[0004] Polyhydroxybutyrate (PHB) is a bacterial polymer with degradability and good biocompatibility. It does not induce inflammation or immune responses in animals or humans, making it widely used in the medical field as a drug delivery carrier, surgical swabs, bandages, and internal fixation splints for fractures. It also has the potential to be processed into tissue engineering scaffold materials for bone defect repair. Zinc oxide is a piezoelectric ceramic with higher piezoelectric properties than polymers, but its brittleness and difficulty in processing limit its application alone in bone tissue repair engineering. Furthermore, studies have shown that high concentrations (60 μg / mL) of zinc oxide nanoparticles more readily reduce the bioactivity of bone marrow mesenchymal stem cells compared to low concentrations (30 μg / mL), indicating concentration-dependent cytotoxicity.

[0005] Electrospinning, also known as electroblotting, is a method of obtaining fibers under electrostatic stretching. This technology can obtain micro- and nano-sized fibers in a relatively simple way. Fiber scaffold materials prepared using electrospinning technology have morphology and structure similar to the extracellular matrix, and its unique production process can easily incorporate functional nanoparticles into polymer fibers, giving it unique advantages in the preparation of tissue engineering scaffolds.

[0006] Aerogels, a type of three-dimensional porous nanofiber material, were first proposed by Professor Kistler in 1931. They possess excellent properties such as high porosity and superior mechanical strength, and can mimic the in vivo growth environment of cells, making them ideal tissue engineering scaffold materials. Currently, there is considerable research on the application of aerogels in wound repair. In vivo, cells are surrounded by other cells, tissues, and the extracellular matrix, an environment significantly different from the two-dimensional culture environment commonly used in laboratories. Much evidence suggests that cells grown in three-dimensional culture systems exhibit more physiological phenotypes, interactions, and responses. Therefore, creating an extracellular matrix-mimicking environment for osteoblasts would be more conducive to bone growth and remodeling. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for preparing piezoelectric nanofiber aerogel, wherein the prepared piezoelectric nanofiber aerogel has a porous structure and good piezoelectric properties, which can accelerate the repair of bone defects.

[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0009] A method for preparing piezoelectric nanofiber aerogel includes the following steps:

[0010] S1. Add zinc oxide to hexafluoroisopropanol, ultrasonically disperse for 25-35 minutes, then add polyhydroxybutyrate, and magnetically stir for 22-26 hours to obtain PHB@ZnO solution;

[0011] S2. The PHB@ZnO solution obtained in step S1 is loaded into an electrospinning device to form a nanofiber membrane by electrospinning. The nanofiber membrane is then placed in a vacuum drying oven and vacuum dried for 24 hours to obtain the PHB@ZnO nanofiber membrane.

[0012] S3. Add the PHB@ZnO nanofiber membrane obtained in step S2 to deionized water, homogenize for 5-15 minutes to obtain a short PHB@ZnO nanofiber dispersion;

[0013] S4. Add chitosan powder to deionized water, stir for 10 minutes, then add glacial acetic acid and continue stirring for 25-35 minutes to obtain a chitosan solution.

[0014] S5. Mix the short PHB@ZnO nanofiber dispersion obtained in step S3, the chitosan solution obtained in step S4, and glutaraldehyde evenly to obtain a mixture. Pour the mixture into a mold and freeze-dry the mold in a freeze dryer for 24 hours to obtain piezoelectric nanofiber aerogel.

[0015] Furthermore, in step S1 of the present invention, the ratio of zinc oxide, hexafluoroisopropanol, and polyhydroxybutyrate is (0.05-0.2) g: (5-10) mL: (0.4-0.8) g.

[0016] Furthermore, in step S2 of the present invention, during electrospinning, the distance between the spinning head and the collector is 18cm, the spinning injection speed is 0.1~0.5mm / min, the receiving speed is 50~100rpm, the positive voltage is 15~18kV, and the negative voltage is -3~-5kV.

[0017] Furthermore, in step S3 of the present invention, the ratio of PHB@ZnO nanofiber membrane to deionized water is (0.2-0.5) g: (10-30) mL.

[0018] Furthermore, in step S4 of the present invention, the ratio of chitosan powder, deionized water, and glacial acetic acid is (2-4) g: 100 mL: (0.5-2) mL.

[0019] Furthermore, in step S5 of the present invention, the volume ratio of short PHB@ZnO nanofiber dispersion, chitosan solution, and glutaraldehyde is (2-7):(3-8):(0.03-0.08), and the freeze-drying temperature is 0°C.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. This invention incorporates ZnO nanoparticles into the biodegradable polymer PHB, and fabricates a nanofiber membrane using electrospinning technology. This membrane is then homogenized to form short nanofibers, which are then cross-linked with chitosan and freeze-dried to autonomously assemble a novel three-dimensional nanofiber aerogel scaffold, namely a piezoelectric nanofiber aerogel. This material possesses a porous structure and excellent piezoelectric properties. It not only structurally mimics the three-dimensional environment of cell growth but also generates charges through external ultrasonic stimulation to simulate the piezoelectric effect of bone, thereby promoting the adhesion and growth of bone marrow mesenchymal stem cells and stimulating bone tissue regeneration, thus accelerating the repair and healing of bone defects. In step S5, chitosan acts as a binder to bond the nanofibers and also serves as a scaffold.

[0022] 2. This invention reduces the cytotoxicity of zinc oxide by encapsulating it with PHB. Compared with autologous bone and allogeneic bone materials, the piezoelectric nanofiber aerogel prepared by this invention has good biocompatibility, reduces the risk of infection, alleviates the patient's pain, and avoids rejection and inflammatory reactions that may be caused by other inorganic materials. Moreover, compared with traditional scaffold materials such as ceramics, the piezoelectric nanofiber aerogel prepared by this invention is easier to process.

[0023] 3. The piezoelectric nanofiber aerogel prepared by this invention is biodegradable and can be replaced by autologous bone tissue in the later stage of bone defect healing, thus sparing patients the pain of having to undergo surgery again to remove the stent.

[0024] 4. The construction of the piezoelectric nanofiber aerogel described in this invention is a new extension of the traditional piezoelectric material structure and provides more options for the clinical treatment of bone defects. Attached Figure Description

[0025] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, do not constitute an undue limitation of the invention. In the drawings:

[0026] Figure 1 This is a scanning electron microscope image of the PHB@ZnO nanofiber membrane prepared in Example 1 of the present invention;

[0027] Figure 2 This is a scanning electron microscope image of the piezoelectric nanofiber aerogel prepared in Example 1 of the present invention;

[0028] Figure 3 The piezoelectric properties of the piezoelectric nanofiber aerogel prepared in Example 1 of this invention and the aerogel prepared in the comparative example are shown in the figure.

[0029] Figure 4 The images show the cell biocompatibility test results of the piezoelectric nanofiber aerogel prepared in Example 1 of this invention and the blank control group.

[0030] Figure 5 The images show the piezoelectric nanofiber aerogel prepared in Example 1 of this invention and the CT images of rat tibial repair in the blank group. Detailed Implementation

[0031] The present invention will now be described in detail with reference to specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0032] Example 1

[0033] Piezoelectric nanofiber aerogels were prepared according to the following steps:

[0034] S1. Add 0.1g of zinc oxide to 5mL of hexafluoroisopropanol, disperse by ultrasonication for 30 minutes, then add 0.6g of polyhydroxybutyrate, and stir magnetically for 24 hours to obtain PHB@ZnO solution;

[0035] S2. The PHB@ZnO solution obtained in step S1 is loaded into an electrospinning device to form a nanofiber membrane by electrospinning. The nanofiber membrane is placed in a vacuum drying oven and vacuum dried for 24 hours to obtain a PHB@ZnO nanofiber membrane. During electrospinning, the distance between the spinning head and the collector is 18 cm, the spinning push speed is 0.1 mm / min, the receiving speed is 50 rpm, the positive voltage is 15 kV, and the negative voltage is -3 kV.

[0036] S3. Add 0.2g of the PHB@ZnO nanofiber membrane obtained in step S2 to 20mL of deionized water, homogenize for 10 minutes to obtain a short PHB@ZnO nanofiber dispersion;

[0037] S4. Add 2g of chitosan powder to 100mL of deionized water, stir for 10 minutes, then add 1mL of glacial acetic acid, and continue stirring for 30 minutes to obtain a chitosan solution.

[0038] S5. The short PHB@ZnO nanofiber dispersion obtained in step S3 with a volume ratio of 6:4:0.04, the chitosan solution obtained in step S4, and glutaraldehyde are mixed evenly to obtain a mixture. The mixture is poured into a mold and the mold is placed in a freeze dryer to freeze dry at 0°C for 24 hours to obtain piezoelectric nanofiber aerogel.

[0039] Example 2

[0040] Piezoelectric nanofiber aerogels were prepared according to the following steps:

[0041] S1. Add 0.1g of zinc oxide to 5mL of hexafluoroisopropanol, disperse by ultrasonication for 30 minutes, then add 0.6g of polyhydroxybutyrate, and stir magnetically for 24 hours to obtain PHB@ZnO solution;

[0042] S2. The PHB@ZnO solution obtained in step S1 is loaded into an electrospinning device to form a nanofiber membrane by electrospinning. The nanofiber membrane is placed in a vacuum drying oven and vacuum dried for 24 hours to obtain a PHB@ZnO nanofiber membrane. During electrospinning, the distance between the spinning head and the collector is 18 cm, the spinning push speed is 0.1 mm / min, the receiving speed is 50 rpm, the positive voltage is 15 kV, and the negative voltage is -3 kV.

[0043] S3. Add 0.2g of the PHB@ZnO nanofiber membrane obtained in step S2 to 20mL of deionized water, homogenize for 10 minutes to obtain a short PHB@ZnO nanofiber dispersion;

[0044] S4. Add 2g of chitosan powder to 100mL of deionized water, stir for 10 minutes, then add 1mL of glacial acetic acid, and continue stirring for 30 minutes to obtain a chitosan solution.

[0045] S5. The short PHB@ZnO nanofiber dispersion obtained in step S3 with a volume ratio of 3:7:0.04, the chitosan solution obtained in step S4, and glutaraldehyde are mixed evenly to obtain a mixture. The mixture is poured into a mold and the mold is placed in a freeze dryer to freeze dry at 0°C for 24 hours to obtain piezoelectric nanofiber aerogel.

[0046] Example 3

[0047] Piezoelectric nanofiber aerogels were prepared according to the following steps:

[0048] S1. Add 0.05g of zinc oxide to 6mL of hexafluoroisopropanol, sonicate for 25 minutes, then add 0.4g of polyhydroxybutyrate, and stir magnetically for 22 hours to obtain a PHB@ZnO solution;

[0049] S2. The PHB@ZnO solution obtained in step S1 is loaded into an electrospinning device to form a nanofiber membrane by electrospinning. The nanofiber membrane is placed in a vacuum drying oven and vacuum dried for 24 hours to obtain a PHB@ZnO nanofiber membrane. During electrospinning, the distance between the spinning head and the collector is 18 cm, the spinning push speed is 0.2 mm / min, the receiving speed is 60 rpm, the positive voltage is 18 kV, and the negative voltage is -5 kV.

[0050] S3. Add 0.3g of the PHB@ZnO nanofiber membrane obtained in step S2 to 10mL of deionized water, homogenize for 5 minutes to obtain a short PHB@ZnO nanofiber dispersion;

[0051] S4. Add 3g of chitosan powder to 100mL of deionized water, stir for 10 minutes, then add 0.5mL of glacial acetic acid, and continue stirring for 25 minutes to obtain a chitosan solution.

[0052] S5. The short PHB@ZnO nanofiber dispersion obtained in step S3 with a volume ratio of 2:3:0.03, the chitosan solution obtained in step S4, and glutaraldehyde are mixed evenly to obtain a mixture. The mixture is poured into a mold and the mold is placed in a freeze dryer to freeze dry at 0°C for 24 hours to obtain piezoelectric nanofiber aerogel.

[0053] Example 4

[0054] Piezoelectric nanofiber aerogels were prepared according to the following steps:

[0055] S1. Add 0.2g of zinc oxide to 10mL of hexafluoroisopropanol, sonicate for 35 minutes, then add 0.8g of polyhydroxybutyrate, and stir magnetically for 26 hours to obtain PHB@ZnO solution;

[0056] S2. The PHB@ZnO solution obtained in step S1 is loaded into an electrospinning device to form a nanofiber membrane by electrospinning. The nanofiber membrane is placed in a vacuum drying oven and vacuum dried for 24 hours to obtain a PHB@ZnO nanofiber membrane. During electrospinning, the distance between the spinning head and the collector is 18 cm, the spinning push speed is 0.5 mm / min, the receiving speed is 100 rpm, the positive voltage is 16 kV, and the negative voltage is -4 kV.

[0057] S3. Add 0.5g of the PHB@ZnO nanofiber membrane obtained in step S2 to 30mL of deionized water, homogenize for 15 minutes to obtain a short PHB@ZnO nanofiber dispersion;

[0058] S4. Add 4g of chitosan powder to 100mL of deionized water, stir for 10 minutes, then add 2mL of glacial acetic acid, and continue stirring for 35 minutes to obtain a chitosan solution.

[0059] S5. The short PHB@ZnO nanofiber dispersion obtained in step S3 with a volume ratio of 7:8:0.08, the chitosan solution obtained in step S4, and glutaraldehyde are mixed evenly to obtain a mixture. The mixture is poured into a mold and the mold is placed in a freeze dryer to freeze dry at 0°C for 24 hours to obtain piezoelectric nanofiber aerogel.

[0060] Comparative Example 1: Chitosan solution, prepared from step S4 of Example 1.

[0061] Comparative Example 2: Similar to Example 1, except that in step S5, the volume ratio of the short PHB@ZnO nanofiber dispersion obtained in step S3 to the chitosan solution obtained in step S4 is 9:1.

[0062] Piezoelectric performance testing

[0063] The test samples were: piezoelectric nanofiber aerogels prepared in Examples 1-2 and Comparative Example 2, and Comparative Example 1. The output performance of the samples was tested using an oscillation excitation system. The test platform consisted of an excitation system and a signal acquisition system, which simulated vibrations in the actual environment and acquired output signals, respectively: a signal generator produced frequency and voltage signals; a power amplifier increased the voltage signal to drive the exciter; and an oscilloscope was used for signal sampling. After connecting the samples to wires, they were implanted into bone defects in rats, and the material was stimulated in vivo using ultrasound to generate piezoelectric signals. The in vivo voltage signal was measured using an oscilloscope.

[0064] Test results are as follows Figure 3 As shown, the voltage signal of Example 1 is the strongest, while the voltage signal of Comparative Example 1 is the weakest, proving that the piezoelectric nanofiber aerogel prepared by the present invention has good piezoelectric properties.

[0065] Biocompatibility testing

[0066] The test sample was a piezoelectric nanofiber aerogel (5 mm in diameter and 3 mm in height) prepared in Example 1. After irradiating the sample under UV light for 12 h, it was soaked in 75% ethanol for 6 h, washed three times with PBS buffer to remove residual ethanol, and then placed in a 24-well plate. 20 μL of cell suspension (1 × 10⁻⁶ cells) was added to each well. 6 (Number of samples). The samples were cultured at 37°C in a 5% CO2 saturated humidified atmosphere for 4 hours, then 1 mL of culture medium was added to soak the samples, and they were cultured again at 37°C in a 5% CO2 saturated humidified atmosphere. Cells were treated with ultrasound stimulation, and the biocompatibility of the samples was tested using the CCK-8 assay.

[0067] Test results are as follows Figure 4 As shown: Figure 4 In each group of bar charts, the left side represents the blank group and the right side represents the sample group. Figure 4 It can be seen that the absorbance of the sample group is higher than that of the blank group, which proves that the piezoelectric nanofiber aerogel prepared by the present invention has good biocompatibility.

[0068] Bone defect repair performance test

[0069] The test sample was the piezoelectric nanofiber aerogel prepared in Example 1. SD rats were anesthetized with 3% sodium pentobarbital, and a cylindrical defect (3 mm in diameter and 3 mm in depth) was drilled in the proximal tibia using a 3 mm drill bit. The sterilized sample was implanted into the defect area, and the muscle and skin were carefully sutured. Ultrasonic stimulation was applied to the defect area twice a week for 10 minutes each time. Two months later, the rats were euthanized with an overdose of pentobarbital, and tibial specimens were collected for μCT to observe the bone defect repair.

[0070] Test results are as follows Figure 5 As shown: Figure 5 The upper half is the blank group, and the lower half is the sample group. Figure 5 It can be seen that the sample group showed good repair, while the blank group showed almost no repair, proving that the piezoelectric nanofiber aerogel prepared in this invention has good bone defect repair performance.

[0071] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for preparing piezoelectric nanofiber aerogel, characterized in that: Includes the following steps: S1. Add zinc oxide to hexafluoroisopropanol, ultrasonically disperse for 25-35 minutes, then add polyhydroxybutyrate, and magnetically stir for 22-26 hours to obtain a PHB@ZnO solution. The ratio of zinc oxide, hexafluoroisopropanol, and polyhydroxybutyrate is (0.05-0.2) g: (5-10) mL: (0.4-0.8) g. S2. The PHB@ZnO solution obtained in step S1 is loaded into an electrospinning device to form a nanofiber membrane by electrospinning. The nanofiber membrane is then placed in a vacuum drying oven and vacuum dried for 24 hours to obtain the PHB@ZnO nanofiber membrane. S3. Add the PHB@ZnO nanofiber membrane obtained in step S2 to deionized water, homogenize for 5-15 minutes to obtain a short PHB@ZnO nanofiber dispersion; S4. Add chitosan powder to deionized water, stir for 10 minutes, then add glacial acetic acid and continue stirring for 25-35 minutes to obtain a chitosan solution. S5. The short PHB@ZnO nanofiber dispersion obtained in step S3 with a volume ratio of 6:4:0.04, the chitosan solution obtained in step S4, and glutaraldehyde are mixed evenly to obtain a mixture. The mixture is poured into a mold, and the mold is placed in a freeze dryer to freeze dry for 24 hours to obtain piezoelectric nanofiber aerogel. The freeze drying temperature is 0℃.

2. The method for preparing a piezoelectric nanofiber aerogel according to claim 1, characterized in that: In step S2, during electrospinning, the distance between the spinning head and the collector is 18cm, the spinning injection speed is 0.1-0.5mm / min, the receiving speed is 50-100rpm, the positive voltage is 15-18kV, and the negative voltage is -3--5kV.

3. The method for preparing a piezoelectric nanofiber aerogel according to claim 1, characterized in that: In step S3, the ratio of PHB@ZnO nanofiber membrane to deionized water is (0.2-0.5) g: (10-30) mL.

4. The method for preparing a piezoelectric nanofiber aerogel according to claim 1, characterized in that: In step S4, the ratio of chitosan powder, deionized water, and glacial acetic acid is (2-4) g: 100 mL: (0.5-2) mL.

Images