Method and application of zirconium-silver-doped bone electroactive osteogenesis-promoting piezoelectric film

Abstract

The invention provides a method and application of a zirconium-silver-doped bone electroactive osteogenesis-promoting piezoelectric film, and belongs to the technical field of material modification. According to the preparation method, zirconium elementary substance and silver elementary substance are used as sputtering sources, and zirconium and silver are sputtered and deposited on a PVDF bone electroactivity square film in an inert gas atmosphere, so that the zirconium-silver doped bone electroactivity osteogenesis promoting piezoelectric film is obtained. The zirconium-silver-doped bone electroactive osteogenesis-promoting piezoelectric film has excellent biocompatibility, antibacterial performance and osteogenesis induction activity, and the problems that in the prior art, a PVDF bone electroactive film is poor in osseointegration and insufficient in bone healing speed and effectiveness are effectively solved; the zirconium-silver-doped bone electroactive osteogenesis-promoting piezoelectric film is suitable for the aspects of tissue engineering, bone defect repair and the like, and the application scene of the PVDF bone electroactive film in the oral cavity and other parts is expanded.

Description

Technical Field

[0001] The invention relates to the technical field of material modification, and in particular to a method and application of a zirconium-silver doped osteoelectrically active osteopromoting piezoelectric film. Background Art

[0002] Electroactive polymer materials have important application value in the fields of biomedicine, energy generation and storage, filtration, sensors and actuators. In addition, the excellent plasticity and flexibility of polymers make them widely used in the field of electroactive films. Among them, PVDF (polyvinylidene fluoride polymer) films are widely used in bone tissue engineering, bone repair, biosensors, drug delivery, etc. due to their good piezoelectric properties and biocompatibility.

[0003] Although PVDF bone electroactive films are widely used in various fields, they still face some challenges and problems in practical applications, such as poor stability of electrical signals and poor induction of bone regeneration. In recent years, zirconium-silver nanomaterials have been shown to have good bioactivity and biocompatibility, and can regulate immune responses. Sputtering coating technology uses ions to bombard the surface of the target material to knock out the atoms of the target material. The deposition of atoms produced by sputtering on the surface of the substrate is called sputtering coating. During sputtering, after the gas is ionized, the gas ions fly to the target material connected to the cathode under the action of the electric field, and the electrons fly to the surface of the substrate to deposit into a thin film. This technology has the advantages of fast film formation speed, uniform film composition, stable film performance, and high film adhesion strength. At present, this technology is mostly used for the preparation of glass films, and the preparation of metal coatings deposited on bioactive substrates is relatively rare. Summary of the invention

[0004] In view of the above shortcomings of the prior art, the present invention provides a method and application of a zirconium-silver doped bone electroactive osteogenic piezoelectric film. The present invention uses magnetron sputtering technology to prepare a zirconium-silver doped bone electroactive osteogenic piezoelectric film with zirconium and silver. The zirconium-silver doped bone electroactive osteogenic piezoelectric film has excellent biocompatibility, antibacterial properties and osteogenic induction activity, which effectively solves the problems of poor bone integration, insufficient bone healing speed and effectiveness of PVDF bone electroactive films in the prior art.

[0005] To achieve the above purpose, the specific technical solutions of the present invention are as follows:

[0006] In a first aspect, the present invention provides a method for preparing a zirconium-silver doped osteoelectrically active osteogenic piezoelectric film, comprising the following steps:

[0007] S1, cutting the PVDF bone electroactive film into square films of appropriate size, cleaning and drying the square films, and storing them in a vacuum for later use;

[0008] S2, sticking a conductive glue on a glass slide, sticking the square film processed in step S1 on the conductive glue, and placing the glass slide upside down into a magnetron sputtering chamber;

[0009] S3. Using zirconium and silver as sputtering sources, in an inert gas atmosphere, zirconium and silver are sputtered and deposited on the PVDF bone electroactive square film to obtain a zirconium-silver doped bone electroactive bone-promoting piezoelectric film.

[0010] Preferably, in step S1, the square film has an area of ​​1 cm x 1 cm-2 cm x 2 cm and a thickness of 28 μm.

[0011] Preferably, in step S3, the gas pressure of the magnetron sputtering is 0.5-5 Pa.

[0012] Preferably, in step S3, the distance between the sputtering source and the square film is 4-10 cm.

[0013] Preferably, in step S3, the sputtering power is 50-100 W.

[0014] Preferably, in step S3, the sputtering temperature is 25-300°C.

[0015] Preferably, in step S3, the inert gas is argon, and the flow rate of the argon is 5-80 sccm.

[0016] Preferably, in step S3, the sputtering time is 10-20 min.

[0017] In a second aspect, the present invention provides a zirconium-silver doped osteoelectroactive osteopromoting piezoelectric film prepared by the method.

[0018] In a third aspect, the present invention provides the use of the zirconium-silver doped osteoelectroactive osteopromoting piezoelectric film in bone defect repair or tissue engineering.

[0019] Compared with the prior art, the present invention is beneficial in that:

[0020] (1) The present invention uses magnetron sputtering technology to prepare a zirconium-silver doped bone electroactive osteogenic piezoelectric film. The zirconium-silver doped bone electroactive osteogenic piezoelectric film has excellent biocompatibility, antibacterial properties and osteoinductive activity, which effectively solves the problems of poor bone integration, insufficient bone healing speed and effectiveness of PVDF bone electroactive films in the prior art; the zirconium-silver doped bone electroactive osteogenic piezoelectric film is suitable for tissue engineering and bone defect repair, and expands the application scenarios of PVDF bone electroactive films in the oral cavity and other parts.

[0021] (2) The present invention effectively utilizes zirconium and silver to modify the PVDF bone electroactive film through magnetron sputtering technology, and the surface of the prepared zirconium-silver doped bone electroactive osteopromoting piezoelectric film has an oxygen content of 23.30wt%, a fluorine content of 2.12wt%, a zirconium content of 56.11wt%, and a silver content of 18.47wt%. The zirconium-silver doped bone electroactive osteopromoting piezoelectric film can effectively enhance the expression of T lymphocyte activation antigen (CD86), interleukin 1β (IL-1β), interleukin 6 (IL-6), interferon regulatory factor 3 (IRF3), TNF receptor associated factor 6 (TRAF6), nuclear factor receptor-κB (NF-κB) and MYD88 by macrophages, thereby exerting excellent antibacterial activity, thereby solving the problem of poor antibacterial performance of PVDF bone electroactive film in the prior art.

[0022] (3) The zirconium-silver doped osteoelectroactive piezoelectric film prepared by the present invention can promote osteoblasts to express RUNX family transcription factor 2 (RUNX2), indicating that it has good osteoinductive activity, solving the problem that the PVDF osteoelectroactive film in the prior art has poor effect in bone bonding and bone repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of the preparation method of the zirconium-silver doped osteoelectroactive osteopromoting piezoelectric film of the present invention;

[0024] Figure 2 The overall imaging of the zirconium-silver doped osteoelectroactive osteopromoting piezoelectric film prepared in Example 1 under a scanning electron microscope;

[0025] Figure 3 The energy spectrum of the zirconium-silver doped osteoelectroactive osteopromoting piezoelectric film prepared in Example 1;

[0026] Figure 4 The relative mRNA expression of CD86 after culturing macrophages RAW264.7 with the films prepared in Example 1 and Comparative Example 1, respectively;

[0027] Figure 5 The relative mRNA expression of IL-1β after culturing macrophages RAW264.7 with the films prepared in Example 1 and Comparative Example 1;

[0028] Figure 6 The relative mRNA expression of IRF3 after culturing macrophage RAW264.7 with the films prepared in Example 1 and Comparative Example 1, respectively;

[0029] Figure 7 The relative mRNA expression of TRAF6 after culturing macrophage RAW264.7 cells with the films prepared in Example 1 and Comparative Example 1, respectively;

[0030] Figure 8 The relative mRNA expression graph of NF-κB after culturing macrophage RAW264.7 with the films prepared in Example 1 and Comparative Example 1 respectively;

[0031] Fig. 9 The relative mRNA expression of IL-6 after culturing macrophages RAW264.7 with the films prepared in Example 1, Comparative Example 2 and Comparative Example 3, respectively;

[0032] Fig.10 The relative mRNA expression of TRAF6 after culturing macrophage RAW264.7 with the films prepared in Example 1, Comparative Example 2 and Comparative Example 3, respectively;

[0033] Fig.11 The relative mRNA expression graph of NF-κB after culturing macrophage RAW264.7 with the films prepared in Example 1, Comparative Example 2 and Comparative Example 3 respectively;

[0034] Fig.12 The relative mRNA expression graph of MYD88 after culturing macrophage RAW264.7 with the films prepared in Example 1, Comparative Example 2 and Comparative Example 3 respectively;

[0035] Fig.13 The relative mRNA expression of RUNX2 after culturing osteoblasts mt-3t3 with the films prepared in Example 1 and Comparative Example 1 respectively; DETAILED DESCRIPTION

[0036] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] The present invention provides a method for preparing a zirconium-silver doped osteoelectrically active osteopromoting piezoelectric film, comprising the following steps:

[0038] S1, cutting the PVDF bone electroactive film into square films of appropriate size, cleaning and drying the square films, and storing them in a vacuum for later use;

[0039] S2, sticking a conductive glue on a glass slide, sticking the square film processed in step S1 on the conductive glue, and placing the glass slide upside down into a magnetron sputtering chamber;

[0040] S3, using zirconium and silver as sputtering sources, in an inert gas atmosphere, sputtering zirconium and silver are deposited on the PVDF bone electroactive square film to obtain a modified zirconium-silver doped bone electroactive bone promoting piezoelectric film. Figure 1 shown.

[0041] In some examples, in step S1, the square film has an area of ​​1 cm x 1 cm-2 cm x 2 cm and a thickness of 28 μm.

[0042] In some examples, in step S3, the gas pressure of the magnetron sputtering is 0.5-5 Pa.

[0043] In some examples, in step S3, the distance between the sputtering source and the square film is 4-10 cm.

[0044] In some examples, in step S3, the sputtering power is 50-100 W.

[0045] In some examples, in step S3, the sputtering temperature is 25-300°C.

[0046] In some examples, in step S3, the inert gas is argon gas, and the flow rate of the argon gas is 5-80 sccm.

[0047] In some examples, in step S3, the sputtering time is 10-20 min.

[0048] Example 1

[0049] A zirconium-silver doped osteoelectrically active osteogenic piezoelectric film, the preparation steps of which are as follows:

[0050] S1. Cut a 28 μm thick PVDF bone electroactive membrane (PolyK, USA) into a 2 cm x 2 cm square film, clean the film with anhydrous ethanol ultrasonically for 15 min, blow dry with nitrogen, and store in a vacuum for later use;

[0051] S2, sticking a conductive glue on a glass slide, sticking the square film processed in step S1 on the conductive glue, and placing the glass slide upside down into a magnetron sputtering chamber;

[0052] S3. Install zirconium and silver in the DC target area of ​​magnetron sputtering, close the chamber and introduce high-purity argon gas to keep the pressure in the chamber at 5 Pa, and deposit zirconium and silver on the square film in an argon atmosphere; wherein, the sputtering distance is 10 cm, the sputtering power is maintained at 100 W, the sputtering temperature is 250 °C, the argon gas flow rate is 5 sccm, and the duration is 15 min, to obtain a zirconium-silver doped osteoelectroactive osteopromoting piezoelectric film.

[0053] The modified zirconium-silver doped PVDF bone electroactive film prepared in Example 1 was imaged as a whole under a magnification of 10,000 times using a scanning electron microscope. Figure 2 As shown. Figure 2 It can be seen that the surface of the modified PVDF bone electroactive film is relatively smooth. The surface element scanning of the obtained modified zirconium-silver doped PVDF bone electroactive film was performed using the energy spectrum scanning module. The results are as follows: Figure 3 As shown in Table 1. Figure 3 As shown in Table 1, stable zirconium and silver are generated on the surface of the modified zirconium-silver doped PVDF bone electroactive film, in which the oxygen content is 23.30wt%, the fluorine content is 2.12wt%, the zirconium content is 56.11wt%, and the silver content is 18.47wt%.

[0054] Table 1: Spectrum scanning specific component results

[0055]

[0056] Example 2

[0057] A zirconium-silver doped osteoelectrically active osteogenic piezoelectric film, the preparation steps of which are as follows:

[0058] S1. Cut a 28 μm thick PVDF bone electroactive membrane (PolyK, USA) into a 2 cm x 2 cm square film, clean the square film with anhydrous ethanol ultrasonically for 15 min, blow dry with nitrogen, and store in a vacuum for later use;

[0059] S2, sticking a conductive glue on a glass slide, sticking the square film processed in step S1 on the conductive glue, and placing the glass slide upside down into a magnetron sputtering chamber;

[0060] S3. Install zirconium and silver in the DC target area of ​​magnetron sputtering, close the chamber and introduce high-purity argon gas to keep the pressure in the chamber at 1 Pa, and deposit zirconium and silver on the square film in an argon atmosphere. The sputtering distance is 10 cm, the sputtering power is maintained at 100 W, the sputtering temperature is 200 °C, the argon flow rate is 5 sccm, and the duration is 20 min to obtain a zirconium-silver doped osteoelectroactive osteopromoting piezoelectric film.

[0061] Example 3

[0062] A zirconium-silver doped osteoelectrically active osteogenic piezoelectric film, the preparation steps of which are as follows:

[0063] S1. Cut a 28 μm thick PVDF bone electroactive membrane (PolyK, USA) into a 2 cm x 2 cm square film. Ultrasonic clean the square film with anhydrous ethanol for 15 min, blow dry with nitrogen and store it in a vacuum.

[0064] S2, sticking a conductive glue on a glass slide, sticking the square film processed in step S1 on the conductive glue, and placing the glass slide upside down into a magnetron sputtering chamber;

[0065] S3. Install zirconium and silver in the DC target area of ​​magnetron sputtering. After closing the chamber, introduce high-purity argon gas to keep the pressure in the chamber at 2.5 Pa. Deposit zirconium and silver on the square film in an argon atmosphere, wherein the sputtering distance is 5 cm, the sputtering power is maintained at 50 W, the sputtering temperature is 100 °C, the argon gas flow rate is 40 sccm, and the duration is 20 min, to obtain a zirconium-silver doped osteoelectrically active osteopromoting piezoelectric film.

[0066] Example 4

[0067] A zirconium-silver doped osteoelectrically active osteogenic piezoelectric film, the preparation steps of which are as follows:

[0068] S1. Cut a 28 μm thick PVDF bone electroactive membrane (PolyK, USA) into a 1 cm x 1 cm square film. Clean the square film with anhydrous ethanol by ultrasonic for 15 min, blow dry with nitrogen and store in a vacuum.

[0069] S2, sticking a conductive glue on a glass slide, sticking the square film processed in step S1 on the conductive glue, and placing the glass slide upside down into a magnetron sputtering chamber;

[0070] S3. Install zirconium and silver in the DC target area of ​​magnetron sputtering, close the chamber and introduce high-purity argon gas to keep the pressure in the chamber at 2.5 Pa, and deposit zirconium and silver on the square film in an argon atmosphere. The sputtering distance is 5 cm, the sputtering power is maintained at 80 W, the sputtering temperature is 25 °C, the argon flow rate is 40 sccm, and the duration is 20 min to obtain a zirconium-silver doped osteoelectroactive osteopromoting piezoelectric film.

[0071] Comparative Example 1

[0072] A PVDF bone electroactive film, the preparation steps of which are as follows:

[0073] A 28 μm thick PVDF bone electroactive membrane (PolyK, USA) was cut into a 2 cm x 2 cm square film, and the film was ultrasonically cleaned with anhydrous ethanol for 15 min and dried with nitrogen to obtain the PVDF bone electroactive membrane.

[0074] Comparative Example 2

[0075] A PVDF bone electroactive film, the preparation steps of which are as follows:

[0076] S1. Cut a 28 μm thick PVDF bone electroactive membrane (PolyK, USA) into a 2 cm x 2 cm square film, clean the film with anhydrous ethanol ultrasonically for 15 min, blow dry with nitrogen, and store in a vacuum for later use;

[0077] S2, sticking a conductive glue on a glass slide, sticking the square film processed in step S1 on the conductive glue, and placing the glass slide upside down into a magnetron sputtering chamber;

[0078] S3. Install zirconium in the DC target area of ​​magnetron sputtering, close the chamber and introduce high-purity argon gas to keep the pressure in the chamber at 5 Pa, and deposit silver on the square film in an argon atmosphere; the sputtering distance is 10 cm, the sputtering power is maintained at 100 W, the sputtering temperature is 250 °C, the argon flow rate is 5 sccm, and the duration is 15 min to obtain a silver-doped osteoelectrically active osteopromoting piezoelectric film.

[0079] Comparative Example 3

[0080] A PVDF bone electroactive film, the preparation steps of which are as follows:

[0081] S1. Cut a 28 μm thick PVDF bone electroactive membrane (PolyK, USA) into a 2 cm x 2 cm square film, clean the film with anhydrous ethanol ultrasonically for 15 min, blow dry with nitrogen, and store in a vacuum for later use;

[0082] S2, sticking a conductive glue on a glass slide, sticking the square film processed in step S1 on the conductive glue, and placing the glass slide upside down into a magnetron sputtering chamber;

[0083] S3. Silver was installed in the DC target area of ​​magnetron sputtering, and high-purity argon gas was introduced after the chamber was closed to keep the pressure in the chamber at 5 Pa. Silver was deposited on the square film in an argon atmosphere. The sputtering distance was 10 cm, the sputtering power was maintained at 100 W, the sputtering temperature was 250 °C, the argon gas flow rate was 5 sccm, and the duration was 15 min. Silver-doped osteoelectrically active osteopromoting piezoelectric film was obtained.

[0084] After soaking the films prepared in Example 1 and Comparative Examples 1-3 in alcohol, they were fully sterilized by ultraviolet irradiation, placed in six-well cell culture plates, and inoculated with macrophages RAW264.7 at a density of 50%. Two days later, when the cell density increased to 80%, the cells were collected for qPCR experiments to detect the expression of T lymphocyte activation antigen (CD86), interleukin 1β (IL-1β), interleukin 6 (IL-6), interferon regulatory factor 3 (IRF3), TNF receptor-associated factor 6 (TRAF6), nuclear factor receptor-κB (NF-κB) and MYD88. Statistical analysis and drawing were performed using graphpad prism software. The results are shown in Figure 2. Figure 4-12 As shown, Figure 4-8 The relative mRNA expression diagrams of CD86, IL-1β, IRF3, TRAF6, and NF-κB after culturing macrophages RAW264.7 with the films prepared in Example 1 and Comparative Example 1, respectively; Figure 9-12 The relative mRNA expression of IL-6, TRAF6, NF-κB, and MYD88 after culturing macrophages RAW264.7 with the films prepared in Example 1, Comparative Example 2, and Comparative Example 3, respectively. Figure 4-12 It can be seen that the PVDF bone electroactive film modified with zirconium and silver can enhance the expression of inflammatory factors such as CD86, IL-1β, IL-6, IRF3, TRAF6, NF-κB and MYD88 in macrophages, indicating that the film can promote the anti-inflammatory activity of macrophages and make them have stronger antibacterial properties.

[0085] After soaking the films prepared in Example 1 and Comparative Example 1 in alcohol, they were fully sterilized by ultraviolet irradiation, placed in six-well cell culture plates, and inoculated with 50% osteoblasts MT-3T3. Two days later, when the cell density increased to 80%, the cells were collected for qPCR experiments to detect the expression of RUNX family transcription factor 2 (RUNX2). Graphpadprism software was used for statistical analysis and drawing. The results are shown in Figure 2. Fig.13 As shown. Fig.13 It can be seen that the PVDF bone electroactive film modified with zirconium and silver can maintain the expression of osteoblast RUNX2 transcription factor, indicating that it has good osteogenic activity.

[0086] Furthermore, the inventors have experimentally verified that the zirconium-silver doped osteoelectroactive osteogenic piezoelectric films prepared in Examples 2-4 all have excellent biocompatibility, antibacterial properties and osteogenic induction activity.

[0087] In summary, the present invention uses zirconium and silver to modify the PVDF bone electroactive film through magnetron sputtering technology, and the prepared zirconium-silver doped bone electroactive osteogenic piezoelectric film has good antibacterial properties and osteogenic activity, which effectively solves the problems of poor bone integration, insufficient bone healing speed and effectiveness of PVDF bone electroactive film in the prior art; the zirconium-silver doped bone electroactive osteogenic piezoelectric film is suitable for tissue engineering and bone defect repair, and expands the application scenarios of PVDF bone electroactive film in the oral cavity and other parts.

[0088] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A method for preparing a zirconium-silver doped osteoelectroactive osteopromoting piezoelectric film, characterized in that: The following steps are involved: S1, cutting the PVDF bone electroactive film into square films of appropriate size, cleaning and drying the square films, and storing them in a vacuum for later use; S2, sticking a conductive glue on a glass slide, sticking the square film processed in step S1 on the conductive glue, and placing the glass slide upside down into a magnetron sputtering chamber; S3. Using zirconium and silver as sputtering sources, in an inert gas atmosphere, zirconium and silver are sputtered and deposited on the PVDF bone electroactive square film to obtain a zirconium-silver doped bone electroactive bone-promoting piezoelectric film.

2. The method for preparing a zirconium-silver doped osteoelectroactive osteopromoting piezoelectric film according to claim 1, characterized in that: The square film has an area of ​​1 cm x 1 cm-2 cm x 2 cm and a thickness of 28 μm.

3. The method for preparing a zirconium-silver doped osteoelectroactive osteopromoting piezoelectric film according to claim 1, characterized in that: The gas pressure of the magnetron sputtering is 0.5-5 Pa.

4. The method for preparing a zirconium-silver doped osteoelectroactive osteopromoting piezoelectric film according to claim 1, characterized in that: The distance between the sputtering source and the square film is 4-10 cm.

5. The method for preparing a zirconium-silver doped osteoelectroactive osteopromoting piezoelectric film according to claim 1, characterized in that: The sputtering power is 50-100W.

6. The method for preparing a zirconium-silver doped osteoelectroactive osteopromoting piezoelectric film according to claim 1, characterized in that: The sputtering temperature is 25-300°C.

7. The method for preparing a zirconium-silver doped osteoelectrically active osteogenic piezoelectric film according to claim 1, characterized in that: The inert gas is argon gas, and the flow rate of the argon gas is 5-80 sccm.

8. The method for preparing a zirconium-silver doped osteoelectroactive osteogenic piezoelectric film according to claim 1, characterized in that: The sputtering time is 10-20 min.

9. A zirconium-silver doped osteoelectroactive osteogenic piezoelectric film prepared by the method according to any one of claims 1 to 8.

10. Use of a zirconium-silver doped osteoelectroactive osteogenic piezoelectric film prepared by the method according to any one of claims 1 to 8 in bone defect repair or tissue engineering.

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