A repair product comprising biodegradable piezoelectric nanofiber membranes, and a preparation method and application thereof

Abstract

The application discloses a repairing product containing biodegradable piezoelectric nanofiber membranes and a preparation method and application thereof. The preparation process is simple, and raw materials are low in price. Compared with pure fiber membranes without doped nanoparticles, piezoelectric polymers in the fiber membranes doped with nanoparticles synergistically promote osteogenesis, and still have biodegradability, meet mechanical performance, and do not affect the piezoelectric performance of the nanofiber membranes.

Description

Technical Field

[0001] This invention belongs to the field of tissue engineering technology, and in particular relates to a repair product comprising a biodegradable piezoelectric nanofiber membrane, its preparation method, and its application. Background Technology

[0002] Regenerative medicine is an emerging interdisciplinary field that offers new hope for treatments including those affecting the nervous system and bone defects. Bone tissue engineering, as an important branch of regenerative medicine, integrates principles of materials science, life sciences, and engineering. The core objective of bone tissue engineering is to develop bioactive materials to repair, regenerate, or replace bone tissue damaged by trauma, disease, tumor resection, or congenital malformations. Since bone is a neurovascularized tissue, neurogenesis is crucial for bone regeneration. Furthermore, bioelectrical phenomena in natural bone play a vital role in bone development and fracture healing, and its intrinsic electric field helps regulate cellular metabolism, such as growth, proliferation, differentiation, and movement. Piezoelectric materials are a class of intelligent functional materials capable of converting mechanical energy into electrical energy. They can generate localized currents within the body, restoring the damaged physiological electrical microenvironment and promoting bone regeneration.

[0003] Existing piezoelectric materials include piezoelectric ceramics, piezoelectric polymers, and piezoelectric composites. Piezoelectric ceramic materials such as lead zirconate titanate and barium titanate have poor biocompatibility and are non-degradable. Piezoelectric polymers such as PVDF and PLLA have poor mechanical properties and weaker piezoelectric performance than piezoelectric ceramics. Piezoelectric composites, obtained by embedding piezoelectric ceramic particles into a polymer matrix, combine the strong piezoelectricity of ceramics with the flexibility of polymers. However, the manufacturing process is complex and costly. Most importantly, the performance of piezoelectric composites largely depends on the quality of the interfacial bonding between the two phases. If the ceramic phase and the polymer matrix are not firmly bonded, stress cannot be effectively transferred to the ceramic phase when the composite is subjected to force, resulting in a reduced output of the positive piezoelectric effect. Conversely, during the inverse piezoelectric effect, the deformation of the ceramic phase cannot be effectively transferred to the matrix, leading to a decrease in the driving displacement. Summary of the Invention

[0004] To address at least some of the technical problems in the prior art, particularly overcoming the drawbacks of piezoelectric ceramics' non-degradability and the defects of piezoelectric polymers' poor mechanical properties and low piezoelectric properties, this invention prepares a composite material incorporating inorganic nanoparticles (e.g., but not limited to magnesium silicate) into a piezoelectric polymer to synergistically promote osteogenic and neurogenic processes, thereby accelerating the bone repair process. Specifically, this invention includes the following:

[0005] In a first aspect, the present invention provides a repair product for tissue damage comprising a biodegradable piezoelectric nanofiber membrane, the piezoelectric nanofiber membrane comprising oriented piezoelectric nanofibers and inorganic nanoparticles, the piezoelectric nanofibers being prepared from raw materials including piezoelectric polymers, the inorganic nanoparticles comprising silicon and magnesium elements, and the doping amount of the inorganic nanoparticles being 2-9 wt% based on the mass of the piezoelectric nanofibers.

[0006] In some embodiments, in the tissue damage repair product according to the present invention, the piezoelectric polymer is selected from polyvinylidene fluoride, polyvinylidene fluoride... Trifluoroethylene copolymer, polyvinylidene fluoride Hexafluoropropylene copolymer, polyvinylidene fluoride At least one of the following: tetrafluoroethylene copolymer, polymethyl methacrylate, polydimethylsiloxane, and L-polylactic acid.

[0007] In some embodiments, the tissue damage repair product according to the present invention includes inorganic nanoparticles comprising magnesium silicate.

[0008] In some embodiments, in the tissue damage repair product according to the present invention, the inorganic nanoparticles have a size of 400-600 nm.

[0009] In some embodiments, the repair product for tissue damage according to the present invention, wherein when a piezoelectric force of 1 N is applied to the surface of the nanofiber membrane at a frequency of 1 Hz to drive the piezoelectricity, the voltage measured by an electrometer is not less than 450 mV.

[0010] In some embodiments, the tissue damage repair product according to the present invention, wherein when measured using a piezoelectric force microscope with an Arrow EFM-20 probe at the tip of an atomic force microscope and an AC voltage at a frequency of 300 kHz, the measured phase curve exhibits a typical 180° phase flip and the amplitude curve exhibits a butterfly shape.

[0011] In some embodiments, in the tissue damage repair product according to the present invention, the piezoelectric nanofiber membrane has a Young's modulus of not less than 50 MPa.

[0012] A second aspect of the present invention provides a method for preparing a repair product for tissue damage, comprising: (1) Dissolve the piezoelectric polymer in an organic solvent to obtain a solution containing the piezoelectric polymer; (2) The inorganic nanoparticles are added to the solution and electrospinned to obtain a piezoelectric nanofiber membrane; (3) Anneal the piezoelectric nanofiber membrane.

[0013] In some embodiments, according to the preparation method of the present invention, the method further includes: (4) a step of ultrasonic treatment of the piezoelectric nanofiber membrane after annealing in step (3).

[0014] A third aspect of the invention provides the use of the piezoelectric nanofiber membrane in the preparation of a repair product for tissue damage.

[0015] The preparation process of this invention is simple and the raw materials are inexpensive. Compared with pure piezoelectric polylactic acid nanofiber membranes, the piezoelectric polymer (such as piezoelectric polylactic acid) in the nanofiber membrane prepared by this invention can synergistically promote osteoogenesis with inorganic nanoparticles (such as magnesium silicate nanoparticles) and still have biodegradability. It meets the mechanical properties without affecting the piezoelectric properties of the nanofiber membrane. Attached Figure Description

[0016] Figure 1 Characterization of the piezoelectric nanofiber membrane of the present invention. Figure A is a scanning electron microscope image, Figure B is an energy dispersive spectroscopy elemental spectrum, and Figure C is the quantitative result of nanofiber diameter.

[0017] Figure 2 Crystal structure (A) and crystallinity (B) of each group of nanofiber membranes of the present invention.

[0018] Figure 3 The piezoelectric properties (A) and piezoelectric response force microscopy measurement results (B, C) of the various groups of nanofiber membranes of the present invention.

[0019] Figure 4 The degradation capacity (A) and ion release curves (B) of each group of nanofiber membranes of the present invention measured by plasma mass spectrometry.

[0020] Figure 5 Stress-strain curves of nanofiber membranes before and after doping (A) and stress-strain curves of nanofiber membranes before and after annealing (B).

[0021] Figure 6 The in vitro evaluation results of the nanofiber membrane of the present invention in promoting osteogenic differentiation. A shows the toxicity test results, and B shows the live / dead cell staining results.

[0022] Figure 7 The proliferation (A) and cell adhesion (B, C) of the nanofiber membrane of the present invention on MC3T3-E1 cells.

[0023] Figure 8 The alkaline phosphatase activity detection results (A) and alizarin red staining results of the nanofiber membrane of the present invention (B).

[0024] Figure 9 Results of the nanofiber membrane of the present invention on the expression of osteogenic-related genes in MC3T3-E1 cells.

[0025] Figure 10 The results of the neural repair assessment of the nanofiber membrane of the present invention. A shows the RSC96 cytotoxicity test results, and B shows the live / dead cell staining results.

[0026] Figure 11 The results of the nanofiber membrane of the present invention on the expression of neurotrophic factor genes in RSC96 cells.

[0027] Figure 12 The antibacterial test results of the nanofiber membrane of the present invention.

[0028] Figure 13 Evaluation results of the nanofiber membrane of the present invention in vivo for repairing skull defects. A is the result of Micro-CT scan, and B is the quantitative result of bone volume / tissue volume (BV / TV), trabecular bone number (Tb.N), and trabecular bone separation (Tb.Sp). Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention.

[0032] A repair product comprising a biodegradable piezoelectric nanofiber membrane In one aspect, the present invention provides a repair product comprising a biodegradable antibacterial piezoelectric nanofiber membrane, the fiber membrane comprising oriented piezoelectric nanofibers and inorganic nanoparticles attached to the surface and / or within the piezoelectric nanofibers, the piezoelectric nanofibers being prepared from raw materials including piezoelectric polymers, the inorganic nanoparticles comprising silicon and magnesium elements, and the doping amount of the inorganic nanoparticles being 2-9 wt% based on the mass of the piezoelectric nanofibers.

[0033] In this invention, directional arrangement refers to the arrangement of individual nanofibers in a parallel or substantially parallel manner. Those skilled in the art will understand that directionally arranged piezoelectric nanofibers can be obtained by controlling the parameters during the electrospinning process. In this invention, the diameter of the nanofibers is generally 500-700 nm.

[0034] In this invention, the piezoelectric nanofibers are prepared from raw materials including piezoelectric polymers. The type of piezoelectric polymer is not particularly limited, and examples include, but are not limited to, polyvinylidene fluoride (PVDF). Trifluoroethylene copolymer, polyvinylidene fluoride Hexafluoropropylene copolymer, polyvinylidene fluoride The piezoelectric nanofibers are selected from at least one of tetrafluoroethylene copolymer, polymethyl methacrylate, polydimethylsiloxane, and polylactic acid (PLA), preferably PLA. In this invention, the molecular weight of the piezoelectric nanofibers is not particularly limited and can vary in the range of 50,000-700,000 Daltons, preferably 10-15 W Daltons.

[0035] In this invention, inorganic nanoparticles are distributed on the surface and / or inside the piezoelectric nanofibers, and contain silicon and magnesium elements. In a preferred embodiment, the inorganic nanoparticles are magnesium silicate nanoparticles. The inorganic nanoparticles of this invention can be prepared by the manufacturer or purchased from commercially available products, and are not particularly limited in this regard. In this invention, the average particle size of the inorganic nanoparticles is not limited, but is preferably 400-600 nm.

[0036] This invention has found that while introducing nanoparticles into L-polylactic acid (PLA) fiber membranes can improve mechanical properties (e.g., tensile strength), particle agglomeration affects directional alignment and is not conducive to crystallization, resulting in a significant decrease in electrical properties. However, by controlling the mass ratio of nanoparticles, an optimal balance between mechanical and electrical properties can be achieved, thus breaking the critical window of the traditional technology's trade-off between "improved mechanical properties inevitably leading to deteriorated electrical properties." In a preferred embodiment of this invention, the doping amount of the inorganic nanoparticles is 3-8 wt% based on the mass of the piezoelectric nanofibers, more preferably 4-7 wt%, even more preferably 4-6 wt%, and most preferably 4.5-5.5 wt%, for example 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, and 5.5 wt%. When the amount added is too low, the mechanical property enhancement effect is insufficient, which is not conducive to repair (such as bone repair and nerve repair); while when the amount added is too high, the nanoparticles are prone to agglomeration, which significantly reduces the electrical properties, thus not conducive to antibacterial and repair (such as bone repair and nerve repair).

[0037] In a preferred embodiment, when a cyclic force of 1 N is applied to the surface of the nanofiber membrane at a frequency of 1 Hz to drive the piezoelectricity, the voltage measured by an electrometer is not less than 450 mV, preferably not less than 500 mV, and even more preferably not less than 600 mV, such as 700, 800, or 900 mV, and particularly not less than 1 V. In a specific embodiment, the output voltage of the undoped nanoparticle fiber membrane is approximately 430 mV, the output voltage of the fiber membrane doped with 1% nanoparticles is approximately 800 mV, the output voltage of the fiber membrane doped with 5% nanoparticles is approximately 1.1 V, and the output voltage of the fiber membrane doped with 10% nanoparticles is approximately 450 mV.

[0038] In a preferred embodiment, when using a piezoelectric response force microscope with an Arrow EFM-20 probe at the tip of an atomic force microscope and applying an AC voltage at a frequency of 300 kHz, the measured phase curve shows a 180° phase reversal, and the amplitude curve shows a butterfly shape, indicating that the material exhibits typical characteristics of a piezoelectric material and has good piezoelectric properties.

[0039] In a preferred embodiment, the Young's modulus of the piezoelectric nanofiber membrane of the present invention is not less than 50 MPa, preferably not less than 55 MPa, for example not less than 60, 70, 80, 90, or 100 MPa. When the nanofiber membrane is not annealed, compared to the Young's modulus of the undoped nanofiber membrane (approximately 45.9 MPa), the Young's modulus of the fiber membrane doped with inorganic nanoparticles is significantly increased (approximately 77.9 MPa), indicating that the doping of inorganic nanoparticles enhances the mechanical strength of the nanofiber membrane. It was also observed that after annealing, although the Young's modulus of both groups of nanofiber membranes increased (approximately 56.9 MPa and approximately 96.5 MPa respectively), the increased fiber crystallinity after annealing led to a decrease in toughness.

[0040] In a preferred embodiment, the piezoelectric nanofiber membrane is a fiber membrane that has undergone annealing and / or ultrasonic treatment. The annealing treatment is a two-stage annealing process, which includes: subjecting the piezoelectric nanofiber membrane to a first annealing treatment at 100-110°C (e.g., 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110°C) for 8-12 hours (e.g., 8, 9, 10, 11, 12 hours), allowing it to cool naturally to room temperature, and then subjecting it to a second annealing treatment at 155-165°C (e.g., 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165°C) for 8-12 hours (e.g., 8, 9, 10, 11, 12 hours), followed by allowing it to cool naturally to room temperature.

[0041] In this invention, the parameters for ultrasonic processing are not particularly limited. Preferably, the effective sound intensity of the ultrasonic waves generated by the ultrasonic device (any suitable ultrasonic equipment known in the art) is 0.20. 2.50 W / cm 2 0.25 W / cm 2 The above, preferably 2.0 W / cm 2 Below, 1.5 W / cm is further preferred. 2 Below, for example, 0.5-1.5 W / cm 2 For example, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 W / cm² 2 The frequency of ultrasound is typically 0.5 GHz. 4 MHz, preferably 0.5-1.5 MHz, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 MHz. In some embodiments, ultrasound can be provided in a pulsed manner, which means providing ultrasound by outputting ultrasound at fixed and / or variable time intervals, or by outputting different ultrasounds within fixed and / or variable time intervals, such as 1-20 min, preferably 1. 15 min, preferably 5-15 min, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 min, etc.

[0042] In the ultrasonic treatment of the present invention, the duty cycle can be controlled within a suitable range. An excessively high duty cycle may cause cells to be dislodged from their cell walls and die. In a preferred embodiment, the duty cycle is not higher than 20%, for example, not higher than 15%, more preferably 5%-12%, for example 5, 6, 7, 8, 9, 10, 11, 12%.

[0043] Preparation method One aspect of the present invention provides a method for preparing a repair product comprising a biodegradable piezoelectric nanofiber membrane, comprising: (1) Dissolve the piezoelectric polymer in an organic solvent to obtain a solution containing the piezoelectric polymer; (2) The inorganic nanoparticles are added to the solution and electrospinned to obtain a piezoelectric nanofiber membrane; (3) Anneal the piezoelectric nanofiber membrane.

[0044] In a preferred embodiment, the preparation method of the present invention further includes: (4) ultrasonic treatment of the piezoelectric nanofiber membrane after annealing in step (3). In a preferred embodiment, the preparation method of the present invention includes: (1) Dissolve L-polylactic acid in an alcohol solution to prepare a solution with a mass-volume ratio of 5-50% (w / v), preferably 5-40%, more preferably 5-30%, and even more preferably 5-20%, for example 10-20%, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20%; (2) Magnesium silicate nanoparticles with a mass concentration of 2-9% (preferably 3-8%, further preferably 4-7%, even more preferably 4-6%, most preferably 4.5-5.5%, such as 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5%) are added to the solution and ultrasonically dispersed, and then electrospinned until a piezoelectric nanofiber membrane with oriented nanofibers is obtained. (3) Anneal the piezoelectric nanofiber membrane.

[0045] application One aspect of the present invention provides the application of piezoelectric nanofiber membranes in the preparation of products for tissue damage repair, preferably bone defect repair products or nerve damage repair products. The specific type of product is not particularly limited and can be an ointment, cream, lotion, gel, paste, etc., or the product for repair can be a dressing, microneedle array / patch, or tissue engineering scaffold. Alternatively, the product can also be a medical device for repair, such as, but not limited to, an ultrasound therapy device comprising the nanofiber membrane of the present invention, wherein the ultrasound therapy device is provided with an ultrasound generating unit configured to generate an effective sound intensity of 0.20. 2.50 W / cm 2 0.25 W / cm 2 The above is preferred, with 2.4 W / cm² being more desirable. 2 Below, for example, 1-1.5 W / cm 2 The frequency is generally 0.5. 4 MHz, preferably 0.5-1.5 MHz ultrasound.

[0046] In this invention, the bone defect repair effect can be confirmed by indicators including but not limited to bone volume / tissue volume (BV / TV), number of trabeculae (Tb.N), trabeculae separation (Tb.Sp), and antibacterial rate, and the corresponding measurement methods are well known in the art.

[0047] In this invention, the repair effect of nerve damage can be confirmed by indicators including but not limited to neurotrophic factors, and the corresponding measurement methods are well known in the art.

[0048] Antibacterial methods One aspect of the present invention provides an antibacterial method comprising the step of using a nanofiber membrane as described herein. In some embodiments, the method is for non-therapeutic purposes, and in other embodiments, the method of the present invention is an in vitro method. Antibacterial effects include inhibiting the growth and reproduction of Gram-positive and / or Gram-negative bacteria.

[0049] Methods to promote the expression of cellular neurotrophic factors One aspect of the present invention provides a method for promoting the expression of cellular neurotrophic factors, comprising the steps of contacting and culturing cells using a nanofiber membrane as described in this invention. In some embodiments, the method is for non-therapeutic purposes, such as drug screening, drug structure optimization, etc. In some embodiments, the method of the present invention is an in vitro method.

[0050] In one specific implementation, the cells are nerve-like cells, particularly RSC96 cells.

[0051] Methods to promote osteogenic gene expression One aspect of the present invention provides a method for promoting osteogenic gene expression in cells, comprising the steps of contacting and culturing cells using a nanofiber membrane as described in this invention. In some embodiments, the method is for non-therapeutic purposes, such as drug screening, drug structure optimization, etc. In some embodiments, the method of the present invention is an in vitro method.

[0052] In one specific implementation, the cells are mouse cranial anterior bone cells (MC3T3-E1 cells).

[0053] Example 1 I. Preparation of Fiber Membranes 70 mL of anhydrous ethanol, ammonia, and deionized water were added to a beaker and stirred for 30 min. Then, 83.332 g of tetraethyl orthosilicate was added to the solution and stirring continued for 4 h. The solution gradually turned milky white and eventually formed a stable colloidal suspension. The suspension was centrifuged at 8000 r / min for 10 min, washed three times with deionized water and ethanol respectively, then centrifuged and dried to obtain the final silica spheres. 0.2 g of the synthesized silica spheres were weighed and placed in a solution containing 0.305 g of magnesium chloride hexahydrate, 1.07 g of ammonium chloride, and 2 mL of ammonia. The mixture was stirred until the colloidal spheres were uniformly dispersed in the mixed solution. The mixture was then transferred to a reaction vessel and subjected to a hydrothermal synthesis reaction for 12 h. After washing and drying, magnesium silicate nanoparticles were obtained.

[0054] Piezoelectric nanofiber membranes were fabricated using electrospinning technology. Specifically, a 16% (w / v) solution of polylactic acid (PLLA) with a molecular weight of 150,000 was dissolved in hexafluoroisopropanol and stirred. Then, magnesium silicate nanoparticles with a mass concentration of 5% were added to the solution and ultrasonically dispersed for 30 min. The prepared solution was placed in a 5 mL syringe fitted with a 22-gauge flat needle and positioned 12 cm away from the collector. The solution flow rate was 1.5 mL / h, the ambient temperature was 25°C, the humidity was 40±15%, the positive voltage was 14 kV, and the negative voltage was 2 kV.

[0055] The nanofiber membrane was placed in a vacuum drying oven overnight to completely evaporate any residual organic solvent. The prepared nanofiber membrane was then placed between two Teflon plates and annealed at 105°C and 160°C for 10 h respectively to enhance their piezoelectric properties.

[0056] As a control, piezoelectric nanofiber membranes with different mass concentrations were prepared using magnesium silicate nanoparticles at mass concentrations of 1% and 10%.

[0057] II. Characterization Piezoelectric composite scaffolds with different magnesium silicate weight ratios, prepared by electrospinning and post-annealing, were named pPLLA / 1MS, pPLLA / 5MS, and pPLLA / 10MS, respectively. The undoped magnesium silicate piezoelectric scaffold was named PA, and the optimal ratio PLLA / 5MS piezoelectric scaffold was named PAMS. Scanning electron microscopy (SEM) showed that the fibers in each group were oriented, and the more doped the fibers, the more nanoparticles were observed. The results are as follows: Figure 1 As shown in Figure A, SEM observation of the magnesium silicate nanoparticles revealed that they exhibited uniformly sized spherical shapes, similar to sea urchin spheres, with a size of 400-600 nm. Energy dispersive spectroscopy (EDS) elemental mapping showed that they were composed of O, Si, and Mg elements. Furthermore, EDS elemental mapping analysis was performed on the nanofiber film, showing that it was composed of C, O, Si, and Mg elements, as shown in Figure A. Figure 1 As shown in B, the nanofiber diameters of PLLA and PLLA / 5MS were evaluated using ImaginJ software. Doping with nanoparticles resulted in a smaller fiber diameter, as shown in Figure B. Figure 1 As shown in C.

[0058] Magnesium silicate nanoparticles and PLLA, PA and PAMS scaffolds were characterized by X-ray diffraction. Figure 2 The result A indicates that the crystal structure of the magnesium silicate nanoparticles has been determined and is consistent with the standard card (JCPDS 02-1390). Figure 2 The results of B indicate that the crystallinity of the PAMS scaffold doped with 5 wt% is improved.

[0059] The piezoelectric properties of each group of nanofiber membranes were characterized using an electrometer. Equal-volume samples were prepared, with single-sided silver conductive strips attached to their top and bottom as electrodes. These electrodes were then connected to the electrometer, and the nanofiber membranes were sealed with polytetrafluoroethylene (PTFE) tape to prevent triboelectric interference. A cyclic force of 1 N was applied to the surface of the nanofiber membrane at a frequency of 1 Hz to drive the piezoelectricity, and the voltage changes of each group of electrometers were observed and recorded. The results showed that the output voltage was highest when doped with 5 wt% magnesium silicate nanoparticles, while the voltage decreased when doped with 10 wt%. This decrease may be due to excessive doping leading to particle agglomeration and affecting the directional alignment of the nanofibers, thus hindering crystallization. Therefore, 5 wt% was considered the optimal doping ratio. Figure 3 As shown in Figure A. When using piezoelectric force microscopy (PFM) for measurements, an Arrow EFM-20 probe was used at the tip of the atomic force microscope, and an AC voltage at a frequency of 300 kHz was applied. The results are as follows: Figure 3 As shown in BC, the PFM test results once again demonstrate that the PAMS nanofiber membrane has good piezoelectric properties. Under the applied bias voltage, the phase curve of the nanofiber membrane exhibits a typical 180° phase flip, and the amplitude curve shows a butterfly shape, indicating that the nanofiber membrane has the typical characteristics of piezoelectric materials.

[0060] The degradation ability of the scaffold was assessed by placing it in PBS solution at 37°C, and the scaffold weight was measured at each time point (1, 3, 7, 14, 21, and 28 days). Compared with the PA scaffold, the PAMS scaffold doped with magnesium silicate nanoparticles showed a faster degradation trend, as shown in the results below. Figure 4 As shown in Figure A. Simultaneously, this example uses inductively coupled plasma mass spectrometry (ICP-MS) to measure the ion concentration in the solution collected at each time point (days 1, 3, 5, and 7). The obtained ion release curves demonstrate that Si... 4+ and Mg 2+ It can be continuously released from the composite stent, with the following results: Figure 4 As shown in B.

[0061] The scaffold was subjected to a uniaxial tensile test in a universal testing machine at a tensile speed of 1 mm / min, and the stress-strain curves were recorded. The mechanical properties of the scaffold doped with magnesium silicate nanoparticles were significantly enhanced, as shown in the results. Figure 5 As shown in Figure A, after annealing, the increased crystallinity of the PA and PAMS supports leads to a decrease in toughness, but an increase in tensile strength, as shown in Figure A. Figure 5 As shown in B.

[0062] Example 2 This embodiment is used to evaluate the in vitro osteogenic differentiation-promoting effect of the piezoelectric composite scaffold prepared in Example 1. This includes the evaluation of the toxicity, proliferation, adhesion, and in vitro osteogenic capacity of the piezoelectric composite scaffold on MC3T3-E1 cells. The toxicity and proliferation were assessed using the CCK8 assay. After co-culturing the scaffolds with MC3T3-E1 cells for 1, 3, and 5 days, the results were detected using a CCK8 assay kit. The results are as follows: Figure 6 As shown in Figure A, the Calcein-AM / PI staining kit was used to stain live and dead cells. No dead cells were observed under the microscope. The results are as follows. Figure 6 As shown in B, cell viability decreased after doping with 10 wt%, and the number of viable cells was reduced under the microscope, thus further proving that 5 wt% is the optimal doping ratio.

[0063] The piezoelectric composite scaffold stimulated by ultrasound after co-culturing for 3 to 5 days significantly promoted cell proliferation, as shown in the following results. Figure 7 As shown in Figure A. Cell adhesion was observed under scanning electron microscopy after double staining with phalloidin and DAPI, followed by gradient dehydration with ethanol. In the control group, cells spread normally. In the PA and PAMS groups, MC3T3-E1 cells were observed to grow along the long axis of the fibers. Cells in all groups spread well. The results are as follows: Figure 7 As shown in BC.

[0064] The evaluation method for osteogenic differentiation capacity of cells involved alkaline phosphatase staining and quantitative analysis, along with Alizarin Red staining and quantitative analysis. Real-time polymerase chain reaction (RT-qPCR) was also used to analyze the mRNA expression of osteogenic-related genes in MC3T3-E1 cells. For alkaline phosphatase staining, MC3T3-E1 cells were co-cultured with and without ultrasound stimulation for 7 days, washed three times with PBS, and stained using an alkaline phosphatase staining kit. A semi-quantitative analysis of alkaline phosphatase activity in MC3T3-E1 cells was performed using an alkaline phosphatase detection kit. The activity of alkaline phosphatase per milligram of protein was calculated by measuring the total protein count in each group using a BCA assay kit. The results are shown below. Figure 8 As shown in Figure A.

[0065] To assess extracellular matrix mineralization, MC3T3-E1 cells were co-cultured with a scaffold for 21 days with and without ultrasound stimulation. Cells were washed three times with PBS, stained with Alizarin Red using an Alizarin Red staining kit, and eluted with calcium nodule elution buffer for quantitative analysis. Results are as follows: Figure 8As shown in B. The method for assessing the expression of osteogenic-related genes was as follows: MC3T3-E1 cells and scaffolds were co-cultured for 7 days with / without ultrasound stimulation, and then total RNA was extracted and reverse transcribed into cDNA. Three osteogenic genes were assessed: Runt-related transcription factor 2 (Runx2), type I collagen (COLⅠ), and osteopontin (OPN). The results are shown in B. Figure 9 As shown.

[0066] Example 3 This embodiment is used to evaluate the neural repair function of the piezoelectric composite scaffold prepared in Example 1, including the toxicity test of the scaffold to RSC96 cells and the evaluation of its in vitro neural repair ability. The toxicity test was performed using the CCK8 assay. After co-culturing each group of scaffolds with RSC96 cells for 1, 3, and 5 days, the results were detected using a CCK8 assay kit. Figure 10 As shown in Figure A, the Calcein-AM / PI staining kit was used to stain live and dead cells. No dead cells were observed under the microscope. The results are as follows. Figure 10 As shown in B.

[0067] For the assessment of neural repair, the expression of neurotrophic factors was analyzed using real-time polymerase chain reaction (RT-qPCR). The assessment method involved co-culturing RSC96 cells and a scaffold for 7 days with and without ultrasound stimulation, followed by extraction of total RNA and reverse transcription into cDNA. Three neurotrophic factor genes were evaluated: brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and nerve growth factor (NGF). The results are as follows: Figure 11 As shown, the results indicate that the piezoelectric composite scaffold of the present invention has significant neurogenesis function, and thus can be used for the recovery of nervous system injuries.

[0068] Example 4 This embodiment is used to test the antibacterial activity of the piezoelectric composite scaffold prepared in Example 1, including its antibacterial activity against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. The evaluation method involves co-culturing the bacterial solution with the scaffold under conditions of with and without ultrasonic stimulation, followed by plate spreading and inverted incubation for 24 hours, followed by observation and photographing. The bacterial survival rate was calculated using the following formula: Bacterial survival rate (%) = CFU1 / CFU0 × 100, where CFU0 is the number of colonies in the blank group, and CFU1 is the number of colonies in different samples. The results are as follows: Figure 12 As shown.

[0069] Example 5 This embodiment is used to evaluate the in vivo repair of skull defects using the piezoelectric composite scaffold prepared in Example 1.

[0070] Thirty male SD rats were randomly divided into six groups for a skull defect model: (A) blank control group; (B) blank control group + US; (C) PA scaffold; (D) PAMS scaffold; (E) PA scaffold + US; (F) PAMS scaffold + US. After intraperitoneal anesthesia, a critical-sized full-thickness bone defect (3 mm in diameter) was established in the skull using a saline-cooled trephine. During the surgery, appropriate samples were placed to cover the defect according to the group, except for the control group which did not receive a sample. The surgical area was then cleaned and the wound sutured. Penicillin was administered three days post-surgery to prevent wound infection, and ultrasound stimulation was performed on the second post-operative day. Ultrasound parameters were set to 1.3 W / cm². 2 Ultrasound stimulation was performed once daily for 10 minutes at a frequency of 1 MHz and a duty cycle of 50% for 14 days. Eight weeks post-surgery, rats were euthanized and their entire skulls were dissected to assess bone repair. The dissected skulls were rinsed with saline and fixed in 4% paraformaldehyde solution for 48 h. Micro-CT scans were used to scan samples from each group, and bone microstructure was measured using three-dimensional reconstruction, including bone volume / tissue volume (BV / TV), trabecular bone number (Tb.N), and trabecular bone separation (Tb.Sp). Results are shown below. Figure 13 As shown in A and B, the results indicate that the piezoelectric composite scaffold prepared in this invention can be used in vivo for the repair of skull defects, and the PAMS scaffold + US has a significant repair effect compared with other groups.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A repair product for tissue damage, characterized in that, The invention includes a biodegradable piezoelectric nanofiber membrane comprising oriented piezoelectric nanofibers and inorganic nanoparticles. The piezoelectric nanofibers are prepared from raw materials including piezoelectric polymers, and the inorganic nanoparticles contain silicon and magnesium elements, with the doping amount of the inorganic nanoparticles being 2-9 wt% based on the mass of the piezoelectric nanofibers.

2. The tissue damage repair product according to claim 1, characterized in that, The piezoelectric polymer is selected from polyvinylidene fluoride and polyvinylidene fluoride. Trifluoroethylene copolymer, polyvinylidene fluoride Hexafluoropropylene copolymer, polyvinylidene fluoride At least one of the following: tetrafluoroethylene copolymer, polymethyl methacrylate, polydimethylsiloxane, and L-polylactic acid.

3. The tissue damage repair product according to claim 1, characterized in that, The inorganic nanoparticles include magnesium silicate.

4. The tissue damage repair product according to claim 1, characterized in that, The inorganic nanoparticles have a size of 400-600 nm.

5. The tissue damage repair product according to claim 1, characterized in that, When a cyclic force of 1 N is applied to the surface of the nanofiber membrane at a frequency of 1 Hz to drive the piezoelectricity, the voltage measured by the electrometer is not less than 450 mV.

6. The tissue damage repair product according to claim 1, characterized in that, When using a piezoelectric force microscope with an Arrow EFM-20 probe at the tip of an atomic force microscope and applying an AC voltage at a frequency of 300 kHz, the measured phase curve shows a 180° phase flip, and the amplitude curve shows a butterfly shape.

7. The tissue damage repair product according to claim 1, characterized in that, The Young's modulus of the piezoelectric nanofiber membrane is not less than 50 MPa.

8. The method for preparing a tissue damage repair product according to any one of claims 1-7, characterized in that, include: (1) Dissolve the piezoelectric polymer in an organic solvent to obtain a solution containing the piezoelectric polymer; (2) The inorganic nanoparticles are added to the solution and electrospinned to obtain a piezoelectric nanofiber membrane; (3) Anneal the piezoelectric nanofiber membrane.

9. The preparation method according to claim 8, characterized in that, Further includes: (4) The step of ultrasonic treatment of the piezoelectric nanofiber membrane after annealing in step (3).

10. The use of the piezoelectric nanofiber membrane according to any one of claims 1-7 in the preparation of a repair product for tissue damage.

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