Ultrasonic-response piezoelectric-conductive integrated nanofiber / hydrogel injectable material for promoting bone regeneration and method

Abstract

The invention discloses an injectable material for promoting bone regeneration through piezoelectric and conductive integrated nanofiber hydrogel with ultrasonic response and a preparation method of the injectable material. The injectable material is composed of piezoelectric and conductive nanofibers and hydrogel. Wherein the piezoelectric conductive nanofiber is prepared from an inorganic piezoelectric material and a piezoelectric polymer through electrostatic spinning, or is prepared from an inorganic / organic composite piezoelectric material and a biological material with conductive performance through electrostatic spinning.

Description

Technical Field

[0001] This invention belongs to the field of hydrogel design, specifically relating to an injectable material and method for promoting bone regeneration using an ultrasonically responsive, piezoelectrically conductive nanofiber / hydrogel. Background Technology

[0002] Repairing bone defects caused by trauma, infection, and other skeletal diseases is a significant clinical challenge that poses a serious threat to patients' daily lives and health. When bone defects are located in inconspicuous areas, making drug administration or surgery difficult, the natural healing process is lengthy, leading to prolonged pain for patients. Inappropriate treatments can further exacerbate bone defects and delay regeneration. Therefore, bone tissue engineering has emerged as a promising therapeutic approach. By rationally designing the structure and composition of biomaterial scaffolds and incorporating various biochemical and biophysical cues to mimic the microenvironment of natural tissues, cellular behavior within engineered structures can be effectively modulated to achieve better recovery. However, challenges such as the mutual constraints of single or multiple cues, as well as adverse reactions like biotoxicity and heterotopic ossification, limit their clinical application. Therefore, developing functional scaffolds that can match the conditions for bone defect repair without relying on active agents is crucial for enhancing bone defect repair.

[0003] The natural bone microenvironment is a layered structure with balanced mechanical, chemical, and electrical properties. Although bone tissue itself is not electroactive, an endogenous electric field (EnEF) exists in living bone, which is believed to be closely related to bone tissue growth, homeostasis, remodeling, and cellular metabolism. The EnEF in bone can be attributed to the piezoelectric effect of its collagen matrix. Conductive materials are primarily used for directly conducting external battery signals, but batteries and wires in contact with tissue increase the risk of post-implantation infection and further amplify tissue damage. Since most human bones are periodically stressed tissues, piezoelectric transducers can be designed to fit this characteristic of bone tissue. Compared to traditional electrochemical transducers (ES), this mechanical-to-electric power supply method offers unique advantages such as non-invasiveness, wireless contact, and self-powered operation. Given this clear need, the advantages of electroactive biomaterials are evident.

[0004] Current electroactive biomaterials mainly include directly conductive biomaterials, piezoelectric biomaterials that convert mechanical force into electrical stimulation, and other responsive biomaterials. Summary of the Invention

[0005] In view of this, the main objective of the present invention is to provide an injectable material and method for promoting bone regeneration using an ultrasonically responsive piezoelectric conductive nanofiber / hydrogel.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0007] An injectable material for promoting bone regeneration by ultrasound-responsive piezoelectric conductive integrated nanofibers / hydrogels, consisting of piezoelectric conductive nanofibers and hydrogels;

[0008] The piezoelectric conductive nanofibers are made by electrospinning inorganic piezoelectric materials and piezoelectric polymers, or by electrospinning inorganic / organic composite piezoelectric materials and biomaterials with conductive properties.

[0009] Preferably, the hydrogel is one of the following: natural polymer-based hyaluronic acid, chitosan, collagen, fibroin, sodium alginate hydrogel, synthetic polymer-based polyethylene glycol, polylactic acid-glycolic acid copolymer, poly(N-isopropylacrylamide), and methacrylic anhydride gelatin hydrogel.

[0010] Preferably, the inorganic piezoelectric material is an inorganic piezoelectric material containing sodium potassium tartrate, diammonium ethylene tartrate, dipotassium tartrate, potassium sulfate, aluminum nitride, zinc oxide, sodium potassium niobate, barium titanate, boron nitride, lead zirconate titanate, modified lead zirconate titanate, lead metaniobate, lithium lead barium niobate, or modified lead titanate.

[0011] The piezoelectric polymer is one of polyvinylidene fluoride, polyvinylidene fluoride-trifluoroethylene copolymer, polylactic acid, polyamide, polypropylene, polyetherketone, and polyphenylene sulfide;

[0012] Preferably, the biomaterial with conductive properties is a conductive polymer, a carbon-based material, or a metal-based material;

[0013] The conductive polymer is one of polypyrrole, polyaniline, and poly(3,4-ethylenedioxythiophene); the carbon-based material is one of graphene and carbon nanotubes.

[0014] The metal-based material is one of gold nanoparticles, silver nanoparticles, or the two-dimensional nanomaterial MXene.

[0015] Preferably, the precursor solution for the piezoelectric conductive nanofibers is dispersed by at least one of an organic solvent, water, and an ionic liquid;

[0016] The organic solvent is at least one of dimethylformamide, dimethylacetamide, tetrahydrofuran, chloroform, and hexafluoroisopropanol, and the ionic liquid is at least one of 1-ethyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium chloride, and 1-ethyl-3-methylimidazolium acetate.

[0017] A method for preparing an injectable material of integrated piezoelectric and conductive nanofibers / hydrogels that promotes bone regeneration in response to ultrasound, the method comprising:

[0018] Conductive nanofibers are prepared by combining inorganic / organic nanomaterials with conductive properties with inorganic / organic polymers with piezoelectric properties.

[0019] Nanofibers with conductive properties are polarized to prepare nanofibers with piezoelectric and conductive functions.

[0020] Short piezoelectric conductive nanofiber rods were prepared from piezoelectric conductive nanofibers.

[0021] Shortened piezoelectric conductive nanofiber rods were added to hydrogels to prepare injectable hydrogels with piezoelectric conductive function.

[0022] Preferably, the step of preparing conductive nanofibers from inorganic / organic nanomaterials and piezoelectric inorganic / organic polymers specifically includes:

[0023] Dissolve organic or inorganic composite piezoelectric materials with piezoelectric properties to obtain a precursor solution;

[0024] A biomaterial with conductive properties is added to the precursor solution and ultrasonically dispersed to obtain a mixed solution;

[0025] The mixed solution was collected using a syringe and added to an electrospinning platform for electrospinning to obtain a piezoelectric conductive nanofiber membrane.

[0026] Preferably, the process of dissolving the organic composite piezoelectric material or inorganic composite piezoelectric material with piezoelectric properties to obtain the precursor liquid specifically includes: dissolving the organic composite piezoelectric material or inorganic composite piezoelectric material with piezoelectric properties in at least one of an organic solvent, water, or an ionic liquid to prepare the precursor liquid; the ultrasonic dispersion conditions of the precursor liquid are: dispersion time of 5-15 min, power of 200-1000 W, and frequency range of 80-200 kHz.

[0027] Preferably, the preparation of nanofibers with piezoelectric and conductive functions by polarization treatment of conductive nanofibers specifically includes: drawing the precursor liquid into a 15ml syringe, transferring the syringe to the propulsion platform of the injection pump for electrospinning, wherein the conditions for electrospinning are: DC voltage 8-20KV, roller speed 100-20000rpm, distance between syringe needle and roller 5-18cm, propulsion speed of injection pump 0.3-1.5ml / h, and humidity controlled at 40±5% at room temperature, thereby obtaining an electrospun nanofiber membrane with conductive properties;

[0028] The electrospun nanofiber membrane is subjected to high-temperature polarization. The polarization process involves annealing at 100℃~105℃ for 12 hours, followed by oven cooling to room temperature. The piezoelectric conductive fiber membrane is then transferred onto a Teflon sheet. Another Teflon sheet weighing 120g is placed on top of the fiber membrane, and the fiber membrane in the interlayer is clamped and subjected to a lateral traction force of 5-50N. The membrane is then heated in an oven at 150℃ for 12 hours. After this process is completed, the oven is turned off and the membrane is cooled to room temperature.

[0029] Preferably, the piezoelectric conductive nanofibers are prepared into short piezoelectric conductive nanofiber rods, specifically including: shearing the piezoelectric conductive nanofibers; dispersing the shredded nanofiber sheets in water or tert-butanol at a scale that is as small as possible; cutting the fiber membrane using an adjustable high-speed homogenizer at a speed of 15,000 to 20,000 rpm to prepare short fiber rods of 300 to 3,000 μm; and freeze-drying the homogenized fiber rods to remove tert-butanol and obtain short piezoelectric conductive nanofibers.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] (1) This invention uses electrospinning to prepare piezoelectric conductive nanofibers as the source of electrical stimulation. Under ultrasonic stimulation, mechanical force can be converted into electrical signals, which can provide an electrical signal source for bone defects. The composite material has conductive properties, which can conduct the electrical signals generated by piezoelectricity to the bone defects, which is beneficial to cell migration and tissue regeneration at the bone defects. (2) This invention uses a biocompatible injectable hydrogel composite material, which can avoid excessive surgery, inconvenient drug administration, and invasive infection problems through injection and ultrasound. (3) The injectable piezoelectric conductive nanofiber hydrogel in this invention has a simple preparation method, good conductivity and electroactivity, good repeatability, and can be used to add active drugs or molecules that need to be released according to application requirements to achieve the drug administration effect. It has great application prospects in bone tissue damage repair. Attached Figure Description

[0032] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0033] Figure 1 The piezoelectric output value of an injectable material of ultrasonic response piezoelectric conductivity integrated nanofiber / hydrogel for promoting bone regeneration under ultrasonic oscillation is shown in the present invention.

[0034] Figure 2 The present invention relates to the effect of an injectable material of ultrasonic-responsive piezoelectric conductive nanofiber / hydrogel on the migration of rat bone marrow mesenchymal stem cells under ultrasonic oscillation.

[0035] Figure 3 The present invention relates to an injectable material of ultrasonic response, piezoelectric conductivity integrated nanofiber / hydrogel that promotes bone regeneration. After being injected into a maxillary bone defect in a rat, the bone defect healed 2 weeks later under ultrasonic vibration. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0037] This invention provides an injectable material for promoting bone regeneration using an ultrasonically responsive piezoelectric conductive nanofiber hydrogel, which is composed of piezoelectric conductive nanofibers and hydrogel.

[0038] The piezoelectric conductive nanofibers are made by electrospinning inorganic piezoelectric materials and piezoelectric polymers, or by electrospinning inorganic / organic composite piezoelectric materials and biomaterials with conductive properties.

[0039] The hydrogel is one of the following: natural polymer-based hyaluronic acid, chitosan, collagen, fibroin, sodium alginate hydrogel, synthetic polymer-based polyethylene glycol, polylactic acid-glycolic acid copolymer, poly(N-isopropylacrylamide), and methacrylic anhydride gelatin hydrogel.

[0040] The inorganic piezoelectric material is an inorganic piezoelectric material containing sodium potassium tartrate, diammonium ethylene tartrate, dipotassium tartrate, potassium sulfate, aluminum nitride, zinc oxide, sodium potassium niobate, barium titanate, boron nitride, lead zirconate titanate, modified lead zirconate titanate, lead metaniobate, lead barium lithium niobate, and modified lead titanate.

[0041] The piezoelectric polymer is one of polyvinylidene fluoride, polyvinylidene fluoride-trifluoroethylene copolymer, polylactic acid, polyamide, polypropylene, polyetherketone, and polyphenylene sulfide;

[0042] The biomaterials with conductive properties are conductive polymers, carbon-based materials, and metal-based materials;

[0043] The conductive polymer is one of polypyrrole, polyaniline, and poly(3,4-ethylenedioxythiophene); the carbon-based material is one of graphene and carbon nanotubes.

[0044] The metal-based material is gold nanoparticles, silver nanoparticles, or one of the two-dimensional nanomaterials MXene.

[0045] The precursor solution for the piezoelectric conductive nanofibers is dispersed by at least one of organic solvent, water, and ionic liquid.

[0046] The organic solvent is at least one of dimethylformamide, dimethylacetamide, tetrahydrofuran, chloroform, and hexafluoroisopropanol, and the ionic liquid is at least one of 1-ethyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium chloride, and 1-ethyl-3-methylimidazolium acetate.

[0047] This invention also provides a method for preparing an injectable material that integrates ultrasonic response, piezoelectric conductivity, and nanofiber / hydrogel to promote bone regeneration. The method includes the following steps:

[0048] Step 101: Prepare conductive nanofibers by combining inorganic / organic nanomaterials with piezoelectric properties with inorganic / organic polymers.

[0049] Specifically, a precursor solution is prepared by dissolving an organic composite piezoelectric material or an inorganic composite piezoelectric material with piezoelectric properties in at least one of an organic solvent, water, or an ionic liquid; the ultrasonic dispersion conditions of the precursor solution are: dispersion time of 5-15 min, power of 200-1000 W, and frequency range of 80-200 kHz.

[0050] The precursor solution was drawn into a 15ml syringe, and the syringe was transferred to the injection pump's propulsion platform for electrospinning. The electrospinning conditions were: DC voltage 8-20KV, roller speed 100-20000rpm, distance between syringe needle and roller 5-18cm, injection pump propulsion speed 0.3-1.5ml / h, and humidity controlled at 40±5% at room temperature. Electrospinned nanofiber membranes with conductive properties were obtained under the above conditions.

[0051] The electrospun nanofiber membrane is subjected to high-temperature polarization. The polarization process involves annealing at 100℃~105℃ for 12 hours, followed by oven cooling to room temperature. The piezoelectric conductive fiber membrane is then transferred onto a Teflon sheet. Another Teflon sheet weighing 120g is placed on top of the fiber membrane, and the fiber membrane in the interlayer is clamped and subjected to a lateral traction force of 5-50N. The membrane is then heated in an oven at 150℃ for 12 hours. After this process is completed, the oven is turned off and the membrane is cooled to room temperature.

[0052] Step 102: Polarize conductive nanofibers to prepare nanofibers with piezoelectric and conductive functions; prepare short piezoelectric conductive nanofiber rods from the piezoelectric conductive nanofibers;

[0053] Specifically, piezoelectric conductive nanofibers are sheared to the smallest possible size. The shredded nanofiber sheets are dispersed in water or tert-butanol, and the fiber membrane is cut using an adjustable high-speed homogenizer at a speed of 15,000–20,000 rpm to prepare short fiber rods of 300–3000 μm.

[0054] Step 103: Add the chopped piezoelectric conductive nanofiber rods to the hydrogel to prepare an injectable hydrogel with piezoelectric conductivity.

[0055] Specifically, the homogenized fiber rods are freeze-dried to remove tert-butanol and obtain short piezoelectric conductive nanofibers.

[0056] The injectable hydrogel contains 0.5%–2% (w / v) hyaluronic acid, 1%–3% (w / v) chitosan, 1%–5% (w / v) collagen, 10–50 mg / ml fibrin, and 1%–3% (w / v) sodium alginate hydrogel. It also contains 5%–20% (w / v) synthetic polymer polyethylene glycol, 10%–30% (w / v) polylactic acid-glycolic acid copolymer, 5%–15% (w / v) poly(N-isopropylacrylamide), and 8–15% (w / v) methacrylic anhydride gelatin. The piezoelectric conductive nanofiber hydrogel contains 0.1%–0.8% (w / v) piezoelectric conductive nanofiber short rods.

[0057] Example

[0058] A method for preparing an injectable material that is an ultrasonically responsive, piezoelectric, and conductive nanofiber hydrogel for promoting bone regeneration, wherein the injectable nanofiber hydrogel is prepared by the following steps:

[0059] (1) Preparation of PLLA and PLLA-MXene (Ti3C2) electrospun nanofibers

[0060] The piezoelectric PLLA membrane was prepared by electrospinning. First, the electrospinning conditions were optimized and determined through orthogonal experiments. 0.8 g of poly-L-lactic acid was added to 10 ml of hexafluoroisopropanol, and the solution was stirred overnight at 60°C on a magnetic stirrer until the PLLA powder was completely dissolved into a transparent solution. The precursor solution was transferred to a 10 ml syringe for electrospinning, with the syringe needle replaced by a 22G flat-tipped injection needle. During electrospinning, the solution flow rate was set to 1 mL / h, the distance between the aluminum foil sheets on the grounded collection roller was 14 cm, the applied voltage was 20 kV, and the humidity was controlled at 40 ± 5% at room temperature. The final electrospinned membrane was dried in a fume hood to remove unreacted solvent.

[0061] The preparation methods and conditions for piezoelectric PLLA-MXene(Ti 3C2) nanofiber membranes are the same as those for PLLA membranes, but the preparation of the electrospinning precursor solution differs slightly. Before electrospinning, a 25 mg / ml MXene(Ti3C2) dispersion is added to the overnight dissolved PLLA electrospinning solution at a volume ratio of 1:4, and ultrasonic dispersion is performed in an ice bath for 15 min to ensure that MXene(Ti3C2) is completely dispersed in the electrospinning PLLA solution before the preparation of the PLLA-MXene(Ti 3C2) electrospinning membrane.

[0062] (2) Piezoelectric treatment of electrospun PLLA and PLLA-MXene (Ti 3C2) nanofiber membranes

[0063] For the piezoelectric treatment of electrospun membranes, thermomechanical processing is performed after obtaining the electrospun nanofiber membrane to improve its piezoelectric properties. The specific steps are as follows: After the PLLA electrospun membrane is prepared, the fiber pad undergoes post-treatment. First, it is annealed at 105℃ for 12 hours, then cooled to room temperature in an oven. Next, the fiber pad is peeled off from the aluminum foil and transferred to a Teflon FEP sheet. Another 120g FEP sheet is placed on top of the fiber film; this interlayer prevents the pad from shrinking. The fiber membrane in the interlayer is clamped and pulled with silver wire, then heated in an oven at 150℃ for 12 hours. After this process, the oven is turned off and cooled to room temperature.

[0064] (3) Preparation of piezoelectric nanofiber hydrogels

[0065] Gelatin methacrylamide (GelMA) hydrogel was synthesized based on previous reports. 20 g of porcine gelatin was added to 200 mL of PBS and stirred on a magnetic stirrer at 60 °C for 1 h until completely dissolved. Then, 16 mL of methacrylic anhydride was slowly added, and the mixture was stirred continuously at 60 °C for 3 h. For the last hour of the final 3 h, 800 mL of PBS was preheated in a water bath at 40-50 °C. The Gel-MA solution generated in step 3 was diluted with the preheated PBS. After mixing the concentrated PBS with the solution, the mixture was rotated at 60 °C for 15 min. A dialysis membrane (MW3500D) was prepared in advance, cut to an appropriate size, and soaked in distilled water for easier handling. The diluted gel was transferred to the dialysis membrane, and dialysis was performed at 40-50 °C for approximately one week. The water was changed 2-3 times daily, with membrane reversed each time. On day 8, the GelMA was filtered using a sterile filter, and the sterilized polymer was transferred to a 50 mL centrifuge tube and stored at -80 °C for one day. Finally, the frozen GelMA was lyophilized.

[0066] The nanofiber membrane was sheared to the smallest possible size, and the shredded nanofiber sheets were dispersed in tert-butanol. The fiber membrane was homogenized at 20,000 rpm using an adjustable high-speed homogenizer to prepare shorter fiber rods. The homogenized fiber rods were then freeze-dried to remove the tert-butanol and obtain short nanofibers.

[0067] Hydrogels containing piezoelectric nanofiber membranes PLLA and PLLA / MXene (Ti 3C2) were prepared by dispersing 0.2% w / v PLLA (or PLLA / MXene (Ti 3C2)), 10% w / v GelMA, and 0.25% w / v photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) in PBS solution. Briefly, 10 mL of PBS was added to a brown flask containing 0.025 g of LAP powder to obtain a 0.25% (w / v) LAP solution. Then, 1 g of GelMA powder and 0.02 g of PLLA or 0.02 g of PLLA / MXene (Ti 3C2) were added to 10 mL of the 0.25% (w / v) LAP solution, and the mixture was then placed in an ultrasonic ice bath. After ultrasonic treatment for 15 minutes, the prepared GelMA / PLLA and Gelma / PLLA-MXene(Ti 3C2) solutions were cured under visible light (405nm) for 30 seconds to obtain piezoelectric hydrogels (GelMA+PLLA or GelMA+PLLA+MXene(Ti 3C2) hydrogels).

[0068] Figure 1 To determine the piezoelectric properties of the hydrogels, piezoelectric hydrogels were assembled into piezoelectric generators. Specifically, two electrodes made of copper sheets (5×5cm) were sandwiched together with the hydrogels and encapsulated with polyimide tape. Holes were drilled at the copper sheet electrodes and silver electrode leads were connected. The exposed silver electrode leads were then reinforced with copper tape. These sensors were subjected to 1MHz ultrasound waves on an ultrasonic therapy device. After the hydrogels polymerized, they formed piezoelectric generators. The piezoelectric output was detected using a RIGOLDS1202Z-E digital oscilloscope. The generators were subjected to periodic vibrations generated by a self-controlled linear motor, and the corresponding voltage output under ultrasonic stimulation was recorded by the oscilloscope. The piezoelectric output values ​​of Gelma, Gelma-PLLA, and Gelma-PLLA-MXene hydrogels were measured using 1MHz ultrasound. The hydrogel with MXene (Ti 3C2) added had a maximum output voltage of 125mV, while the hydrogel scaffold with only PLLA added had an output voltage of 33.7mV peak-to-peak.

[0069] (4) Effects of piezoelectric materials under ultrasound stimulation on the migration of bone marrow mesenchymal stem cells (BMSCs)

[0070] Because cell migration experiments require scratching, and hydrogel matrix materials are too soft, extracts were chosen as the culture medium to observe cell migration. Specifically, Gelma, Gelma-PLLA, and Gelma-PLLA-MXene were sterilized, immersed in serum-free culture medium, and incubated at 37°C for 24 hours. The extracts were then collected and co-cultured with BMSC cells to observe cell morphology and growth. The initial cell seeding density for the scratch test was 2.5 × 10⁶, and the serum concentration of the culture medium was 10%. After 24 hours of starvation, when the cells reached 80% confluence, uniform scratches were performed using a pipette tip. The entire experiment used serum-free culture medium and the corresponding extracts, and cell migration was calculated after 24 hours of treatment. Figure 2 As can be seen, there was no significant difference in migration between the surface control group and the Gelma group in the scratch test results. However, both the Gelma-PLLA and Gelma-PLLA-MXene groups significantly improved the cell coverage area. The cell migration rate of the Gelma-PLLA-MXene group was close to 60%, indicating that piezoelectric nanofiber hydrogels can induce BMSC migration under ultrasonic stimulation.

[0071] (5) Repair of alveolar bone defects by piezoelectric conductive hydrogel under ultrasonic stimulation

[0072] After purchasing the animals, they were acclimatized for one week. Once adapted to their environment, 15 male Sprague-Dawley rats (220–250g) were divided into three groups: Control, GelMA-PLLA, and GelMA-PLLA-MXene. All surgeries were performed under general anesthesia, with intraperitoneal injection of 3% sodium pentobarbital. A 2×2×2 cm maxillary bone defect was created approximately 1 cm above the top of the first premolars on both sides using a low-speed dental drill. Control, GelMA-PLLA, and GelMA-PLLA-MXene were injected into the periodontal defects and light-cured for 30 seconds. The wounds were sutured with surgical sutures. The rats were fed eggs and feed for the first week. Those who underwent surgery without any additional materials were designated the Blank group. The maxillary bone defect animal models were subjected to ultrasound treatment after surgery. Ultrasound was performed on the right side of each rat defect, but not the left. A 40 kHz dental ultrasonic device was used for ultrasound treatment, with 15 minutes of stimulation per day.

[0073] Four weeks post-surgery, rats were euthanized, and their entire heads were removed. After tissue trimming, the heads were placed in 4% paraformaldehyde. Bone regeneration at the defect site was analyzed using a 20 μm resolution micro-CT microscope (Quantum GX2 PerkinElmer). Representative sections were obtained from the sagittal plane to observe bone growth. Figure 3 As shown.

[0074] Four weeks after implantation, microscopic CT imaging and 3D reconstruction were performed to observe new bone growth in the bone defect. Figure 3 More newly regenerated and infiltrated bone was observed at the defect margins in the Gelma-P and Gelma-PM2 groups, while no significant new bone formation was observed in the control group. Furthermore, representative sagittal planes also showed greater bone regeneration in the piezoelectric nanofiber groups, especially the Gelma-PM2 group.

[0075] In the sham surgery group, little new bone formation was observed in either the ultrasound (US+) or non-ultrasound (US-) group. In the Gelma-P group, the amount of new bone in the US+ group was greater than that in the US- group, indicating that the Gelma-P piezoelectric material converts ultrasound stimulation into an electrical signal, thereby promoting new bone formation. In the Gelma-PM2 group, the bone defect in the US+ group almost completely healed, indicating that the Gelma-PM2 nanofiber hydrogel has excellent osteogenic function.

[0076] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention.

Claims

1. An ultrasound-responsive piezoelectrically conductive integrated nanofiber / hydrogel bone-regeneration-promoting injectable material, characterized in that, The piezoelectric conductive nanofiber is made of inorganic piezoelectric material and piezoelectric polymer through electrospinning, or made of inorganic / organic composite piezoelectric material and biological material with conductive performance through electrospinning. The piezoelectric conductive nanofiber is made of inorganic piezoelectric material and piezoelectric polymer through electrospinning, or made of inorganic / organic composite piezoelectric material and biological material with conductive performance through electrospinning.

2. The ultrasound-responsive, piezoelectrically conductive integrated nanofiber / hydrogel bone regenerative-promoting injectable material according to claim 1, wherein, The hydrogel is one of natural polymer-based hyaluronic acid, chitosan, collagen, fibrin, sodium alginate hydrogel, synthetic polymer-based polyethylene glycol, polylactic acid-glycolic acid copolymer, poly N-isopropyl acrylamide, and methacrylic anhydride gelatin hydrogel.

3. The ultrasound-responsive, piezoelectrically conductive integrated nanofiber / hydrogel bone-regenerative injectable material of claim 1, wherein, The inorganic piezoelectric material is inorganic piezoelectric material potassium sodium tartrate, ethylenediammonium tartrate, dipotassium tartrate, potassium sulfate, aluminum nitride, zinc oxide, potassium sodium niobate, barium titanate, boron nitride, lead zirconate titanate, modified lead zirconate titanate, lead metaniobate, lead barium lithium niobate, and modified lead titanate. The piezoelectric polymer is one of polyvinylidene fluoride, polyvinylidene fluoride-trifluoroethylene copolymer, polylactic acid, polyamide, polypropylene, polyether ketone, and polyphenylene sulfide.

4. The ultrasound-responsive, piezoelectrically conductive integrated nanofiber / hydrogel bone-regenerative injectable material of claim 1, wherein, The biological material with conductive performance is one of conductive polymer, carbon-based material, and metal-based material. The conductive polymer is one of polypyrrole, polyaniline, and poly(3,4-ethylenedioxythiophene); the carbon-based material is one of graphene and carbon nanotube; and the metal-based material is one of gold nanoparticles, silver nanoparticles, and two-dimensional nanomaterial MXene. The precursor solution of the piezoelectric conductive nanofiber is dispersed by at least one of organic solvent, water, and ionic liquid.

5. The ultrasonic response piezoelectric conductive integrated nanofiber / hydrogel bone regeneration-promoting injectable material according to claim 1, wherein, The organic solvent is at least one of dimethylformamide, dimethylacetamide, tetrahydrofuran, chloroform, and hexafluoroisopropanol; and the ionic liquid is at least one of 1-ethyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium chloride, and 1-ethyl-3-methylimidazolium acetate. The preparation method comprises the following steps:

6. A method for the preparation of the injectable bone regenerative material of the piezoelectrically conductive integrated nanofiber / hydrogel in response to ultrasound waves according to any one of claims 1-5, characterized by, The inorganic / organic nanomaterial with conductive performance and the inorganic / organic polymer with piezoelectric performance are prepared into nanofiber with conductive performance; The nanofiber with conductive performance is polarized to prepare nanofiber with piezoelectric and conductive performance; The piezoelectric conductive nanofiber is prepared into short piezoelectric conductive nanofiber rod; The short piezoelectric conductive nanofiber rod is added into the hydrogel to prepare injectable hydrogel with piezoelectric and conductive performance. The preparation of the nanofiber with conductive performance from the inorganic / organic nanomaterial with conductive performance and the inorganic / organic polymer with piezoelectric performance comprises the following steps:

7. The production method according to claim 6, wherein The organic composite piezoelectric material or inorganic composite piezoelectric material with piezoelectric performance is dissolved to obtain a precursor solution; The biological material with conductive performance is added into the precursor solution and ultrasonically dispersed to obtain a mixed solution; The mixed solution is collected by a syringe and electrospun on an electrospinning platform to obtain a piezoelectric conductive nanofiber membrane. ​ 8. The preparation method according to claim 7, characterized in that, The process of dissolving an organic composite piezoelectric material or an inorganic composite piezoelectric material with piezoelectric properties to obtain a precursor solution specifically includes: dissolving the organic composite piezoelectric material or the inorganic composite piezoelectric material with piezoelectric properties in at least one of an organic solvent, water, or an ionic liquid to prepare a precursor solution; the ultrasonic dispersion conditions of the precursor solution are: dispersion time of 5-15 min, power of 200-1000 W, and frequency range of 80-200 kHz.

9. The preparation method according to claim 7, characterized in that, The process of polarizing conductive nanofibers to prepare nanofibers with piezoelectric and conductive functions specifically includes: drawing the precursor solution into a 15ml syringe, transferring the syringe to the propulsion platform of an injection pump for electrospinning. The electrospinning conditions are: DC voltage 8-20KV, roller speed 100-20000rpm, distance between syringe needle and roller 5-18cm, injection pump propulsion speed 0.3-1.5ml / h, and humidity controlled at 40±5% at room temperature. Electrospinned nanofiber membranes with conductive properties are obtained under the above conditions. The electrospun nanofiber membrane is subjected to high-temperature polarization. The polarization process involves annealing at 100℃~105℃ for 12 hours, followed by oven cooling to room temperature. The piezoelectric conductive fiber membrane is then transferred onto a Teflon sheet. Another Teflon sheet weighing 120g is placed on top of the fiber membrane, and the fiber membrane in the interlayer is clamped and subjected to a lateral traction force of 5-50N. The membrane is then heated in an oven at 150℃ for 12 hours. After this process is completed, the oven is turned off and the membrane is cooled to room temperature.

10. The method of claim 9, wherein, The preparation of short piezoelectric conductive nanofiber rods involves: shearing the piezoelectric conductive nanofibers; dispersing the shredded nanofiber sheets in water or tert-butanol at a scale that is as small as possible; cutting the fiber membrane using an adjustable high-speed homogenizer at a speed of 15,000–20,000 rpm to prepare short fiber rods of 300–3000 μm; and freeze-drying the homogenized fiber rods to remove tert-butanol and obtain short piezoelectric conductive nanofibers.

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