A biomimetic piezoelectric scaffold for promoting bone repair, its preparation method and application

Abstract

This invention relates to the field of biomedical technology, specifically to a biomimetic piezoelectric scaffold for promoting bone repair, its preparation method, and its applications. The biomimetic piezoelectric scaffold provided by this invention is produced by electrospinning PLLA into oriented nanofibers, and then modifying the fiber surface with nano-hydroxyapatite. To enhance the adhesion of hydroxyapatite to the fiber surface, a polydopamine layer is modified between the two as a connecting layer, ultimately resulting in a biomimetic piezoelectric scaffold with high porosity, good osteointegration, and osteogenic promoting effects. This scaffold exhibits good bone regeneration promoting effects in in vitro MSC recruitment, osteogenic differentiation experiments, and repair of skull bone defects in rats.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a biomimetic piezoelectric scaffold for promoting bone repair, its preparation method, and its application. Background Technology

[0002] While acute or small fractures often heal without surgery, large bone defects require orthopedic surgery, posing a significant challenge for both patients and orthopedic surgeons. Currently, there are three main methods of bone grafting: autologous, allogeneic, and artificial scaffold transplantation. Many studies are attempting to develop synthetic materials for bone repair, including polymers, hydrogels, ceramics, and metal scaffolds.

[0003] Based on the structure of bone and the conditions required for bone regeneration, ideal bone graft biomaterials need to meet requirements such as good osteointegration, osteoinductivity, biodegradability, porosity, and biocompatibility. Furthermore, as early as 1954, Japanese scientist Yasuda first reported the piezoelectric effect of bone, where an electric charge is generated on its surface when compressed, with the amount of charge proportional to the pressure. Subsequently, it was discovered that the endogenous electric field generated by this piezoelectric effect in vivo has a significant impact on the bone regeneration process. Therefore, to better promote bone regeneration, piezoelectric bone implant materials have become a major research focus. Common piezoelectric materials include piezoelectric polymers and piezoelectric ceramics. While piezoelectric ceramics possess good piezoelectric properties, they are brittle, difficult to process, and have poor osteointegration. Therefore, piezoelectric polymers, with their better flexibility and processability, have a better application prospect in bone implant materials. Common piezoelectric polymers include polyvinylidene fluoride (PVDF) and its copolymers, poly-L-lactic acid (PLLA), 3-hydroxybutyrate, and other natural polymers (cellulose, collagen, etc.). Compared to natural polymers, synthetic polymers offer advantages such as tunable mechanical properties and lower cost. PVDF, in particular, exhibits high piezoelectric output, but its non-degradability limits its application in implants. Poly-L-lactic acid (PLLA), on the other hand, is degradable, non-toxic, non-irritating, and possesses tunable mechanical properties, making it an important biodegradable polymer material. It has been approved for clinical use by the U.S. Food and Drug Administration. However, its piezoelectric coefficient is lower than that of polyvinylidene fluoride (PVDF), and it suffers from poorer osteointegration and osteogenic induction. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the primary objective of this invention is to provide a method for preparing a biomimetic piezoelectric scaffold for promoting bone repair. This method utilizes PLLA membranes to prepare bone repair scaffold materials with high piezoelectric properties and good bone integration and osteoinductive properties.

[0005] A second objective of this invention is to provide a biomimetic piezoelectric scaffold for promoting bone repair prepared by the above method, which has high piezoelectric properties and meets the requirements of good bone integration and osteoinductive properties.

[0006] A third objective of this invention is to provide the application of the above-mentioned biomimetic piezoelectric scaffold in the preparation of drugs or medical devices that promote bone repair, exhibiting a good bone regeneration promoting effect.

[0007] To achieve the above objectives, the technical solution provided by this invention is as follows:

[0008] According to a first aspect of the present invention, the present invention provides a method for preparing a biomimetic piezoelectric scaffold for promoting bone repair, comprising the following steps:

[0009] Step (1): Provide a poly-L-lactic acid nanofiber membrane with piezoelectric properties and oriented fiber arrangement;

[0010] Step (2): Provide a liquid containing dopamine, and place the poly-L-lactic acid nanofiber membrane described in step (1) into the liquid containing dopamine. The dopamine undergoes self-polymerization on the fiber surface to obtain a polydopamine-modified nanofiber membrane.

[0011] Step (3): Provide a liquid in which hydroxyapatite is dispersed, place the polydopamine-modified nanofiber membrane described in step (2) into the liquid in which hydroxyapatite is dispersed, and further modify the surface of the polydopamine-modified nanofiber membrane with hydroxyapatite to obtain the biomimetic piezoelectric support.

[0012] Furthermore, the preparation process of the poly-L-lactic acid nanofiber membrane in step (1) includes the following steps:

[0013] Step (11): Using high molecular weight poly-L-lactic acid as raw material, poly-L-lactic acid nanofiber membranes with oriented fiber arrangement are prepared by electrospinning.

[0014] Step (12): Anneal the poly-L-lactic acid nanofiber membrane from step (11) to obtain a poly-L-lactic acid nanofiber membrane with piezoelectric properties and oriented fiber arrangement.

[0015] And / or, the poly-L-lactic acid nanofiber membrane described in step (1) has a piezoelectric coefficient greater than 0.5 Pc / N; preferably, it has a piezoelectric coefficient greater than 1 pC / N.

[0016] Furthermore, in step (11), the high molecular weight poly-L-lactic acid has a molecular weight of 300,000 to 500,000.

[0017] Further, the electrospinning method described in step (11) is as follows: the spinning uses a positive high voltage of 8kV to 13kV and a negative high voltage of 2kV to 5kV; the spinning distance is 8cm to 15cm, and the solution extrusion speed is 0.5ml / L to 1.5ml / h;

[0018] And / or, the receiving device used in electrospinning is a high-speed rotating roller with a rotation speed of 800 r / min to 2000 r / min.

[0019] Furthermore, the annealing temperature in step (12) is 120℃~150℃, and the annealing time is 4h~8h.

[0020] Furthermore, in step (2), the self-polymerization process of dopamine on the fiber surface is carried out under the first ultrasonic treatment;

[0021] And / or, the modification process of hydroxyapatite in step (3) is carried out under a second ultrasonic treatment.

[0022] Further, the liquid containing dopamine in step (2) is prepared by the following method: preparing a Tris solution with a pH of 8 to 8.5, and then dispersing dopamine hydrochloride into the Tris solution at a concentration of 2 mg / ml;

[0023] And / or, the first ultrasonic treatment uses an ultrasonic power of 80W to 100W, an ultrasonic frequency of 20kHz to 60kHz, and an ultrasonic treatment time of 0.5h to 2h.

[0024] Further, the preparation method of the liquid containing hydroxyapatite in step (3) is to disperse nano-hydroxyapatite in water at a concentration of 1 mg / ml to 10 mg / ml;

[0025] And / or, the second ultrasonic treatment uses an ultrasonic power of 80W to 100W, an ultrasonic frequency of 20kHz to 60kHz, and an ultrasonic treatment time of 0.5h to 2h.

[0026] According to a second aspect of the present invention, the present invention provides a biomimetic piezoelectric scaffold for promoting bone repair, the scaffold being prepared according to the method described above.

[0027] According to a third aspect of the present invention, the present invention provides the application of the above-described biomimetic piezoelectric scaffold in the preparation of drugs or medical devices that promote bone repair.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] 1. The method for fabricating the biomimetic piezoelectric scaffold provided by this invention involves first preparing a poly-L-lactic acid (PLLA) nanofiber membrane with piezoelectric properties and oriented fiber arrangement, and then modifying the fiber surface with hydroxyapatite (HA). To enhance the adhesion of HA to the fiber surface, a polydopamine (PDA) layer is modified between the two as a connecting layer. Furthermore, the surface of the piezoelectric PLLA nanofiber membrane is more prone to generating charges, attracting microparticles and thus facilitating the modification of its surface with polydopamine and HA. Finally, a biomimetic piezoelectric scaffold with high piezoelectric properties, good osteointegration, and osteoinductive properties is obtained.

[0030] 2. The biomimetic piezoelectric scaffold preparation method provided by the present invention utilizes the fact that the surface of piezoelectric PLLA fibers is more likely to generate charges under ultrasonic action, attracting dopamine to contact the fibers more quickly and form hydrogen bonds with them. Dopamine accumulates on the fiber surface in a short time to form a polydopamine (PDA) layer, thus obtaining PDA-PLLA. This method can control the modification time within 2 hours, which is beneficial to shorten the preparation time and production cost of the scaffold.

[0031] 3. The biomimetic piezoelectric scaffold provided by this invention can rapidly recruit endogenous MSCs to migrate to the defect site in the early stage, and then promote osteogenic differentiation under the action of ultrasonic piezoelectricity, thereby achieving a programmed bone regeneration effect. The biomimetic structure and high porosity of this HPP scaffold have good bone integration, which can induce the directional arrangement of cells and is more conducive to guiding bone tissue to grow into natural bone.

[0032] 4. The raw materials PLLA, PDA and HA used in the biomimetic piezoelectric stent provided by the present invention have good biocompatibility and bioabsorbability, and can eventually be degraded in the body without toxic side effects on organisms.

[0033] 5. The biomimetic piezoelectric scaffold provided by this invention has good application prospects as a preparation of drugs or medical devices that promote bone repair. Experimental results show that in in vitro MSC recruitment, osteogenic differentiation test and repair of rat skull bone defects, the HPP scaffold can recruit MSC migration, significantly promote osteogenic differentiation and promote bone regeneration.

[0034] Instruction manual illustrations

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1SEM images of PLLA, PDA / PLLA, and HPP;

[0037] Figure 2 Photo of the actual HPP film;

[0038] Figure 3 Diagrams showing the diameter distribution of PLLA, PDA / PLLA, and HPP fibers;

[0039] Figure 4 Results of hydrophilicity tests for PLLA, PDA / PLLA, and HPP;

[0040] Figure 5 The results are from PLLA, PDA / PLLA, and HPP stress-strain tests.

[0041] Figure 6 The test results are for the piezoelectric coefficients of PLLA-A and PLLA.

[0042] Figure 7 The piezoelectric performance test results for PLLA-A and HPP;

[0043] Figure 8 Results of PLLA, TCP, and HPP biocompatibility tests;

[0044] Figure 9 A schematic diagram of a scheme for MSCs recruitment testing;

[0045] Figure 10 Staining images of migrating cells in the blank group, PLLA group, and HPP group;

[0046] Figure 11 for Figure 10 The corresponding cell count chart;

[0047] Figure 12 Results of in vitro osteogenic differentiation induction experiments for TCP, PLLA, and HPP groups;

[0048] Figure 13 HE staining images of the skulls of rats in the blank group, PLLA group, and HPP group;

[0049] Figure 14 CT images of skull repair in the blank group, PLLA and HPP group;

[0050] Figure 15 Statistical graphs of new bone area in the blank group, PLLA and HPP group. Detailed Implementation

[0051] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] This invention provides a method for preparing a biomimetic piezoelectric scaffold for promoting bone repair, comprising the following steps:

[0053] Step (1): Provide a poly-L-lactic acid nanofiber membrane with piezoelectric properties and oriented fiber arrangement;

[0054] Step (2): Provide a liquid containing dopamine, and place the poly-L-lactic acid nanofiber membrane described in step (1) into the liquid containing dopamine. The dopamine undergoes self-polymerization on the fiber surface to obtain a polydopamine-modified nanofiber membrane.

[0055] Step (3): Provide a liquid in which hydroxyapatite is dispersed, place the polydopamine-modified nanofiber membrane described in step (2) into the liquid in which hydroxyapatite is dispersed, and further modify the surface of the polydopamine-modified nanofiber membrane with hydroxyapatite to obtain the biomimetic piezoelectric support.

[0056] In the above technical solution, a poly-L-lactic acid nanofiber membrane with piezoelectric properties and oriented fiber arrangement is used as the basic material of the scaffold. Hydroxyapatite (HA) is modified onto the fiber surface. To enhance the adhesion of HA to the fiber surface, a polydopamine (PDA) layer is modified between the two as a connecting layer, ultimately resulting in a biomimetic piezoelectric scaffold with high porosity, good osteointegration, and osteoinductive properties. This scaffold has a structure that mimics natural bone. The HA coating on the outermost layer of the poly-L-lactic acid nanofiber membrane can effectively attract endogenous mesenchymal stem cells (MSCs) to migrate to the scaffold in the early stages, generating piezoelectric signals to stimulate osteogenic differentiation of MSCs and promote bone tissue repair. The oriented fiber structure can induce osteocytes to grow along the fiber structure, which is more conducive to the orientation of regenerated bone tissue formation, thus more closely resembling the structure of natural bone tissue.

[0057] Furthermore, the piezoelectric properties of PLLA nanofiber membranes make it easier to generate charges on their surface, attracting microparticles and facilitating the modification of their surface with polydopamine and hydroxyapatite. The raw materials used in this scaffold, PLLA, PDA, and HA, exhibit good biocompatibility and bioabsorbability, and are ultimately degraded in vivo without toxic side effects.

[0058] Some analytical theories have indicated that the piezoelectricity of poly-L-lactic acid (PLLA) nanofiber membranes is caused by the asymmetric distribution of their molecular chains. Due to differences in raw materials, preparation methods, and preparation parameters, the piezoelectric properties of PLLA nanofiber membranes can vary significantly. Some methods produce amorphous PLLA fiber membranes or membranes with low crystallinity; these untreated PLLA fiber membranes have low or negligible piezoelectric properties. As a further limitation, the PLLA nanofiber membrane with piezoelectric properties described in this invention has a piezoelectric coefficient greater than 0.5 pc / N; more preferably, it has a piezoelectric coefficient greater than 1 pc / N.

[0059] The present invention provides a biomimetic piezoelectric scaffold that utilizes the piezoelectric properties of poly-L-lactic acid (PLLA) nanofiber membranes. It should be noted that the larger the molecular weight of PLLA and the better the fiber orientation, the better the piezoelectric performance. The nanofiber membrane is then subjected to high-temperature annealing to improve the crystallinity of the polymer, ultimately obtaining a PLLA nanofiber membrane with piezoelectric properties. As a preferred embodiment, the present invention provides the following method for preparing a PLLA fiber membrane with high piezoelectric properties, comprising the following steps:

[0060] Step (11): Using high molecular weight poly-L-lactic acid as raw material, poly-L-lactic acid nanofiber membranes with oriented fiber arrangement are prepared by electrospinning.

[0061] Step (12): Anneal the poly-L-lactic acid nanofiber membrane from step (11) to improve the crystallinity of the polymer and obtain a poly-L-lactic acid nanofiber membrane with piezoelectric properties and oriented fiber arrangement.

[0062] While a higher molecular weight is beneficial for improving the piezoelectric properties of PLLA, the higher the molecular weight, the more difficult the spinning process becomes. In a preferred embodiment, in step (11), the high molecular weight poly-L-lactic acid has a molecular weight of 300,000 to 500,000. For example, its molecular weight can be 300,000, 350,000, 400,000, 450,000, or 500,000. As a further preferred embodiment, this invention, through exploration of the spinning process, proposes the following suitable spinning conditions: specifically, spinning uses a positive high voltage of 8kV to 13kV and a negative high voltage of 2kV to 5kV. For example, the positive high voltage can be 8kV, 10kV, 12kV, or 13kV, and the negative high voltage... The high voltage can be 2kV, 3kV, 4kV, and 5kV, etc.; the spinning distance is 8cm to 15cm, and the solution extrusion speed is 0.5ml / L to 1.5ml / h. For example, the spinning distance can be 8cm, 10cm, 12cm, and 15cm. As a further preferred embodiment, the receiving device used for electrospinning is a high-speed rotating roller with a rotation speed of 800r / min to 2000r / min. For example, the rotation speed can be 800r / min, 1000r / min, 1200r / min, 1500r / min, 1800r / min, and 2000r / min. In a preferred embodiment, the annealing temperature is 120℃ to 150℃, and the annealing time is 4h to 8h. For example, the annealing temperature can be 120℃, 130℃, 140℃, and 150℃, and the annealing time can be 4h, 5h, 6h, 7h, and 8h.

[0063] In the above-mentioned technical solution of the present invention, the preparation of a polydopamine layer on the PLLA surface can serve as a connecting bridge to connect HA to the PLLA fiber. The applicant discovered through research that when the above-mentioned poly-L-lactic acid nanofiber membrane with piezoelectric properties is placed in a liquid containing dopamine, the piezoelectric PLLA fiber generates surface charge under the action of ultrasound, attracting dopamine to contact the fiber more quickly and form hydrogen bonds with it. Dopamine accumulates on the fiber surface in a short time to form a polydopamine (PDA) layer, resulting in PDA-PLLA. Compared with the general method of modifying dopamine under shaking conditions, this method shortens the modification time from 24 hours to within 2 hours. As a preferred embodiment, the self-polymerization process of dopamine on the fiber surface in step (2) of the present invention is carried out under the first ultrasonic treatment; and / or, the modification process of hydroxyapatite in step (3) is carried out under the second ultrasonic treatment. In a preferred embodiment, the first and second ultrasonic treatments can each independently employ the following methods, using an ultrasonic power of 80W to 100W, an ultrasonic frequency of 20kHz to 60kHz, and an ultrasonic treatment time of 0.5h to 2h. For example, the ultrasonic frequency can be 80W, 85W, 90W, 95W, and 100W; the ultrasonic frequency can be 20kHz, 30kHz, 40kHz, 50kHz, and 60kHz; and the ultrasonic treatment time can be 0.5h, 1h, 1.5h, and 2h.

[0064] As a preferred embodiment, the present invention provides a preferred method for preparing a liquid containing dopamine, specifically as follows: preparing a Tris solution with a pH of 8 to 8.5, and then dispersing dopamine hydrochloride into the Tris solution at a concentration of 2 mg / ml.

[0065] The scaffold in this invention utilizes the connecting effect of PDA to modify HA onto the outermost side of the fibrous membrane, possessing a natural bone-inspired structure. Compared to existing methods that co-spin HA and PLLA to prepare scaffolds, where the internal HA molecules require PLLA degradation before release, the scaffold in this invention allows for rapid HA release after implantation. This facilitates increased local calcium ion concentration, effectively attracting endogenous mesenchymal stem cells (MSCs) to migrate to the scaffold early on. The resulting piezoelectric signal stimulates osteogenic differentiation of MSCs, promoting bone tissue repair and better leveraging the osteogenic activity-enhancing effect of HA. As a preferred embodiment, the preparation method of the liquid containing hydroxyapatite involves dispersing nano-hydroxyapatite in water at a concentration of 1 mg / ml to 10 mg / ml.

[0066] The biomimetic piezoelectric support prepared by this invention can be processed in terms of thickness and shape according to the actual application scenario. It can also extend the spinning time to increase the film thickness or use gas foaming technology to process the film into a three-dimensional shape.

[0067] Another embodiment of the present invention provides a biomimetic piezoelectric scaffold for promoting bone repair, wherein the scaffold is prepared by the above method. The prepared biomimetic piezoelectric scaffold has a structure that mimics natural bone, possesses high piezoelectric properties, high porosity, good osteointegration, and osteogenic promoting effect. Nano-hydroxyapatite particles are coated on the surface of poly-L-lactic acid nanofibers, which can effectively attract endogenous mesenchymal stem cells (MSCs) to migrate to the scaffold in the early stages, generating piezoelectric signals to stimulate osteogenic differentiation of MSCs, promoting bone tissue repair. Furthermore, the scaffold has good biocompatibility and bioabsorbability, and ultimately degrades in vivo without toxic side effects on the organism.

[0068] Another embodiment of the present invention provides the application of the above-mentioned biomimetic piezoelectric scaffold in the preparation of drugs or medical devices that promote bone repair. The present invention provides experimental verification of the biomimetic piezoelectric scaffold in in vitro MSC recruitment and osteogenic differentiation experiments and in the repair of skull bone defects in rats. Experimental results show that the HPP scaffold can recruit MSC migration, significantly promote osteogenic differentiation, and has a promoting effect on bone regeneration.

[0069] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0070] Example 1: Fabrication of a biomimetic piezoelectric support

[0071] (1) Medical grade PLLA powder with a molecular weight of 350,000 was dissolved in hexafluoroisopropanol solution at a concentration of 10%. After stirring overnight, electrospinning was performed. The spinning conditions were: positive high voltage of 10kV, negative high voltage of 3kV, spinning distance of 10cm, solution extrusion speed of 1ml / h, receiving device of high-speed rotating drum at a rotation speed of 1800r / min (the fibers obtained at this receiving speed are oriented, which is beneficial to improving the piezoelectric properties of the fiber membrane), and spinning time of 90min. After spinning, the obtained film was placed in a vacuum drying oven at room temperature overnight to fully evaporate the residual organic solvent in the film. The obtained film was named PLLA-A film.

[0072] (2) The PLLA-A film was annealed at 140℃ for 8 hours in an oven to improve the crystallinity of PLLA and thus improve the piezoelectric properties of the PLLA film. The annealed PLLA film (named PLLA film) was obtained. The microstructure of the obtained fiber film is as follows: Figure 1 a and Figure 1 The scanning electron microscope (SEM) image of d is shown, where, Figure 1 a is the surface structure of the PLLA film, which shows that the PLLA film has a very large porosity and the fibers are oriented. Figure 1 d is a SEM cross-sectional image of the film, showing that the fiber diameter is between 400 nm and 600 nm;

[0073] (3) Prepare a Tris solution with a pH of 8–8.5. Dissolve dopamine hydrochloride in this solution at a concentration of 2 mg / ml. Place the annealed PLLA film in this solution and ultrasonically bathe it for 90 min at a power of 100 W and a frequency of 40 kHz. Under the action of ultrasound, the piezoelectric PLLA fibers generate surface charges, attracting dopamine to contact the fibers more quickly and form hydrogen bonds with them. Dopamine accumulates on the fiber surface in a short time to form a polydopamine (PDA) layer, thus obtaining PDA / PLLA. Compared with the general method of modifying dopamine under shaking conditions, this method shortens the modification time from 24 h to within 2 h. The microstructure of the obtained PDA / PLLA film is as follows: Figure 1 The scanning electron microscope (SEM) images of b and 1e are shown, where, Figure 1 b is a SEM image of the surface morphology of the PDA / PLLA film. Figure 1 e is a SEM cross-sectional image of the PDA / PLLA film, showing that the polydopamine layer thickness is around 48 nm.

[0074] (4) Weigh nano-hydroxyapatite (hexagonal crystal form, particle size about 10 nm) powder and disperse it in an aqueous solution at a concentration of 5 mg / ml. Disperse the powder thoroughly in an ultrasonic water bath for 10 min. Immerse the PDA / PLLA film obtained in the previous step into this solution, and then continue ultrasonication at a power of 100 W and a frequency of 40 kHz for 1 h. The hydroxyapatite layer is modified. After air drying at room temperature, the scaffold HA / PDA / PLLA (i.e., HPP) is obtained. The microstructure of the obtained HPP scaffold is shown below. Figure 1 The scanning electron microscope (SEM) images of c and 1f are shown, where, Figure 1 c is a surface SEM image of the scaffold, showing that hydroxyapatite particles are uniformly coated on the fiber surface. Figure 1 f is a SEM cross-sectional image of the scaffold, showing that the thickness of the modified layer is approximately 70 nm. A photograph of the prepared HPP film is shown below. Figure 2 As shown.

[0075] Based on the SEM images of fibers in different regions, ImageJ software was used to analyze the fiber diameter of the PLLA film, PDA / PLLA film, and HA / PDA / PLLA film. The results are as follows: Figure 3As shown, the fiber diameter generally increases sequentially with the modification layer, and the final fiber diameter of the HPP scaffold is mostly distributed in the range of 500nm to 800nm.

[0076] The surface hydrophilicity of the above-mentioned PLLA film, PDA / PLLA film, and HA / PDA / PLLA film was tested using a contact angle meter. The hydrophilicity test results are as follows: Figure 4 As shown in the figure. The results showed that the hydrophilicity of the scaffold surface was greatly improved after modification with PDA and HA. This was due to the hydrophilicity of PDA itself and the increase in surface roughness of the scaffold by HA nanoparticles, which is beneficial to cell adhesion and growth. HA, as the main inorganic component of bone, also promotes osteogenic differentiation of MSCs. The synergistic effect of these two factors will greatly improve the osseointegration of the scaffold.

[0077] The stress-strain tests were performed on the above-mentioned PLLA film, PDA / PLLA film, and HA / PDA / PLLA film using a thin film stress tester. The obtained stress-strain curves are shown below. Figure 5 As shown, the Young's modulus of the scaffold gradually increases during the modification process.

[0078] Example 2: Testing the effect of annealing on improving the piezoelectric properties of PLLA

[0079] Experiments were conducted using the unannealed PLLA-A film and the annealed PLLA film from Example 1 above. A gold electrode was deposited on both sides of the film using magnetron sputtering. Then, the piezoelectric coefficient in the D14 direction of the film was measured using a D14 piezoelectric meter. The piezoelectric coefficient test results are as follows: Figure 6 As shown.

[0080] The results showed that the piezoelectric coefficient of PLLA-A without annealing was close to 0 pC / N, while the piezoelectric coefficient of PLLA after annealing reached 2.22 pC / N, indicating that the annealing process effectively improved the piezoelectricity of PLLA.

[0081] Example 3: Piezoelectric Performance Test

[0082] The piezoelectric properties of HPP in Example 1 were tested, with the low-piezoelectric PLLA-A film (unannealed) from Example 1 used as a blank control. A gold electrode layer was deposited on both sides of PLLA-A or HPP using magnetron sputtering, and connected to the positive and negative terminals of an oscilloscope via copper wires. Then, an ultrasonic probe was used to apply a power of 1 W / cm² to the support. 2 The ultrasonic and piezoelectric performance test results are as follows: Figure 7 As shown.

[0083] The results showed that the electrical signal output of the PLLA-A without annealing was almost zero, indicating that it had almost no piezoelectricity. The voltage signal of the HPP bracket was about 210mV, which confirmed the piezoelectricity of the HPP bracket.

[0084] Example 4: Biocompatibility Test

[0085] Biocompatibility testing was performed on the HPP scaffold of Example 1, with cell culture plates (TCP) and the PLLA film without HA modification in Example 1 serving as controls. Primary rat MSCs were seeded on the surface of the culture plates, PLLA films, or HPP scaffolds. CCK8 assays were performed on the cells on days 1, 3, and 5 (the absorbance values ​​obtained were directly proportional to cell viability). Figure 8 The absorbance of the obtained CCK8 supernatant at a wavelength of 450 nm is the result.

[0086] The results showed that the HPP scaffold exhibited excellent biocompatibility and effectively promoted cell proliferation.

[0087] Example 5: MSCs recruitment test

[0088] MSC recruitment tests were performed on the HPP scaffold of Example 1, with blank and unmodified HA PLLA membranes as controls, according to... Figure 9 The schematic diagram of the test protocol shows that a PLLA membrane or HPP scaffold is placed in the lower chamber of the Transwell, and MSCs are seeded in the upper chamber. After 12 hours, the cells on the membrane at the bottom of the upper chamber are stained with crystal violet to observe the number of cells that have migrated from the upper chamber to the membrane. Figure 10 These are staining images of migrating cells in each group. Figure 11 yes Figure 10 Corresponding cell count statistics;

[0089] The results showed that the HPP scaffold could recruit MSCs to migrate, presumably because the HA modified on the HPP scaffold slowly releases calcium ions into the solution, thereby promoting MSC migration.

[0090] Example 6: In vitro promoting effect on osteogenic differentiation

[0091] Osteogenic differentiation induction experiments were performed on the HPP scaffold prepared in Example 1. Cell culture plates and unmodified HA PLLA membranes were used as controls. MSCs were seeded on their surfaces, and after adhesion, osteogenic differentiation medium was used for osteogenic differentiation induction. Cells were subjected to ultrasound stimulation for 2 minutes daily using an ultrasound probe at a power of 1 W / cm². 2 On days 3 and 7, ALP was extracted and quantified from the cells using an alkaline phosphatase (ALP) extraction kit according to its specifications. ALP is a characteristic enzyme in osteoblasts and can reflect the degree of osteogenic differentiation. The test results are as follows: Figure 12 As shown, where, Figure 12 a is a statistical graph of ALP quantification; under the same culture conditions, on day 7, cells in each group were subjected to immunofluorescence staining for BMP2 and OCN (osteoblast-associated protein, indicating the degree of osteogenic differentiation), and then the fluorescence intensity was statistically analyzed. Figure 12 b and Figure 12 c);

[0092] The results showed that the HPP scaffold could significantly promote osteogenic differentiation under the action of ultrasound.

[0093] Example 7: Recruitment of endogenous cells in vivo

[0094] A vertical incision of approximately 1 cm was made on the scalp of the rat. The connective tissue of the skull was separated to expose the skull. Avoiding the midline of the skull, a circular bone defect was created on the right side using a trephine drill, thus obtaining a skull bone defect model in SD rats. The defect area was a circle with a diameter of 5 mm. Blank and unmodified PLLA were used as controls. PLLA or HPP scaffolds were filled into the defect, and the wound was then sutured. Ten days post-surgery, the rat skull was harvested, fixed, decalcified, and stained with hematoxylin and eosin (HE). Figure 13 The result obtained was from HE staining.

[0095] As can be seen, a large number of cells in the HPP group have migrated to the scaffold site and grown into the scaffold, which verifies the cell recruitment effect of HPP, consistent with the results of in vitro experiments (Example 5).

[0096] Example 8: The promoting effect of in vivo scaffold on bone regeneration

[0097] The surgical procedure was the same as in Example 7. The scaffold was allowed to recruit endogenous cells within 10 days post-surgery. From day 11 to day 30, the defect site was treated daily with 0.8 W / cm² of treatment. 2 The rats were subjected to ultrasound stimulation for 2 minutes. Twelve weeks post-surgery, skull fragments were harvested and fixed, followed by CT scans. Figure 14 These are CT images showing the skull repair status of each group. Figure 15 This is a statistical chart of the area of ​​newly regenerated bone.

[0098] As can be seen, at 12 weeks post-surgery, while the control group still had a large number of defect areas, the bone defect areas of the HPP group rats were almost completely healed, verifying the promoting effect of ultrasound-guided HPP scaffold on bone regeneration.

[0099] 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 preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features, all of which should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A method for the preparation of a biomimetic piezoelectric scaffold for promoting bone repair, characterized by, Includes the following steps: Step (1): Provide a poly-L-lactic acid nanofiber membrane with piezoelectric properties and oriented fiber arrangement, wherein the piezoelectric coefficient of the poly-L-lactic acid nanofiber membrane is greater than 0.5 pC / N; Step (2): Provide a liquid containing dopamine, place the poly(L-lactic acid) nanofiber membrane described in step (1) into the liquid containing dopamine, and allow dopamine to self-polymerize on the fiber surface under ultrasonic conditions to obtain a polydopamine-modified nanofiber membrane. Step (3): Provide a liquid in which hydroxyapatite is dispersed, place the polydopamine-modified nanofiber membrane described in step (2) into the liquid in which hydroxyapatite is dispersed, and further modify the surface of the polydopamine-modified nanofiber membrane with hydroxyapatite under ultrasonic conditions to obtain the biomimetic piezoelectric support. The preparation process of the poly-L-lactic acid nanofiber membrane in step (1) includes the following steps: Step (11): Using high molecular weight poly-L-lactic acid as raw material, poly-L-lactic acid nanofiber membranes with oriented fiber arrangement are prepared by electrospinning. Step (12): Anneal the poly-L-lactic acid nanofiber membrane from step (11) to obtain a poly-L-lactic acid nanofiber membrane with piezoelectric properties and oriented fiber arrangement.

2. The preparation method according to claim 1, characterized in that, The poly-L-lactic acid nanofiber membrane described in step (1) has a piezoelectric coefficient greater than 1 pC / N.

3. The preparation method according to claim 2, characterized in that, In step (11), the high molecular weight poly-L-lactic acid has a molecular weight of 300,000 to 500,000.

4. The preparation method according to claim 2 or 3, characterized in that, The electrospinning method described in step (11) is as follows: the spinning uses a positive high voltage of 8kV to 13kV and a negative high voltage of 2kV to 5kV; the spinning distance is 8cm to 15cm and the solution extrusion speed is 0.5ml / h to 1.5ml / h; the receiving device used for electrospinning is a high-speed rotating roller with a rotation speed of 800r / min to 2000r / min.

5. The preparation method according to claim 2 or 3, characterized in that, The annealing temperature in step (12) is 120℃~150℃, and the annealing time is 4h~8h.

6. The preparation method according to claim 1, characterized in that, The liquid containing dopamine in step (2) is prepared by the following method: a Tris solution with pH of 8 to 8.5 is prepared, and then dopamine hydrochloride is dispersed in the Tris solution at a concentration of 2 mg / ml; under the ultrasonic conditions, the ultrasonic power is 80 W to 100 W, the ultrasonic frequency is 20 kHz to 60 kHz, and the ultrasonic treatment time is 0.5 h to 2 h.

7. The preparation method according to claim 1, characterized in that, The preparation method of the liquid containing hydroxyapatite in step (3) is as follows: nano-hydroxyapatite is dispersed in water at a concentration of 1 mg / ml to 10 mg / ml; under the ultrasonic conditions, the ultrasonic power is 80 W to 100 W, the ultrasonic frequency is 20 kHz to 60 kHz, and the ultrasonic treatment time is 0.5 h to 2 h.

8. A biomimetic piezoelectric scaffold for promoting bone repair, characterized in that, The stent is prepared by the method described in any one of claims 1 to 7.

9. The use of the biomimetic piezoelectric scaffold of claim 8 in the preparation of drugs or medical devices that promote bone repair.

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