Method and application of hydroxyapatite and silver composite coating modified antibacterial film

Abstract

The invention provides a method and application of a hydroxyapatite and silver composite coating modified antibacterial film, and belongs to the technical field of material modification. According to the preparation method, silver elementary substances and hydroxyapatite crystals are used as sputtering sources, in the inert gas atmosphere, the hydroxyapatite and silver composite coating is prepared on the surface of a PVDF bone electroactive square film through the magnetron sputtering technology, and the antibacterial promoting film modified by the hydroxyapatite and silver composite coating is obtained. The hydroxyapatite and silver composite coating modified antibacterial promoting film has excellent biocompatibility, antibacterial performance and osteogenesis inducing 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 hydroxyapatite and silver composite coating modified antibacterial 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 for modifying an antibacterial film by using a hydroxyapatite and silver composite coating and application thereof. Background Art

[0002] Bone tissue repair and regeneration is a complex process that requires multiple physiological signals, including biochemical, electrical and mechanical signals, which work together to ensure functional recovery. The inherent piezoelectric properties of bone tissue can convert mechanical stimulation into electrical effects, playing an important role in bone maturation, remodeling and reconstruction. The use of electroactive materials to prepare bone repair scaffolds can simulate the physiological electrical microenvironment of bone tissue, thereby promoting bone regeneration and reconstruction.

[0003] PVDF (polyvinylidene fluoride polymer) bone electroactive film has excellent piezoelectric properties and a large specific surface area. It can effectively simulate the collagen fiber structure in the natural extracellular matrix and instantly generate surface charge when undergoing small mechanical deformation. Previous studies have shown that nanotopography and electrical signals in the extracellular microenvironment are crucial for bone regeneration. Nanostructures can regulate cell behavior through mechanical transmission, thereby inducing bone marrow mesenchymal stem cells (BMSC) to differentiate into osteoblasts. Therefore, PVDF bone electroactive film has a wide range of applications in bone tissue engineering, bone repair, biosensors, drug delivery, etc.

[0004] Although PVDF bone electroactive films have shown great potential, they still face some challenges and problems in practical applications, such as poor electrical signal stability, poor bone regeneration regulation, slow bone healing, etc. Therefore, it is of great clinical significance to design a bone electroactive film with good biocompatibility, antibacterial activity, stability, and the ability to regulate bone regeneration. Summary of the invention

[0005] In view of the deficiencies of the above prior art, the present invention provides a method and application of a hydroxyapatite and silver composite coating modified antibacterial film. The present invention adopts magnetron sputtering technology to modify the antibacterial film using a hydroxyapatite and silver composite coating. The antibacterial film modified by the hydroxyapatite and silver composite coating has excellent biocompatibility, antibacterial properties and osteoinduction 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.

[0006] To achieve the above purpose, the specific technical solutions of the present invention are as follows:

[0007] In a first aspect, the present invention provides a method for modifying an antibacterial film by a hydroxyapatite and silver composite coating, comprising the following steps:

[0008] 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;

[0009] 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;

[0010] S3. Using silver and hydroxyapatite crystals as sputtering sources, in an inert gas atmosphere, silver and hydroxyapatite are sputtered and deposited on the PVDF bone electroactive square film to obtain an antibacterial film modified by a hydroxyapatite and silver composite coating.

[0011] 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.

[0012] Preferably, in step S3, the gas pressure of the magnetron sputtering is 0.5-5 Pa.

[0013] Preferably, in step S3, the distance between the sputtering source and the square film is 4-10 cm.

[0014] Preferably, in step S3, the sputtering power is 50-100 W.

[0015] Preferably, in step S3, the sputtering temperature is 25-300°C.

[0016] Preferably, in step S3, the inert gas is argon gas, and the flow rate of the argon gas is 5-80 sccm.

[0017] Preferably, in step S3, the sputtering time is 10-20 min.

[0018] In a second aspect, the present invention provides an antibacterial film modified by a hydroxyapatite and silver composite coating prepared by the method.

[0019] In a third aspect, the present invention provides the use of the antibacterial film modified by the hydroxyapatite and silver composite coating in bone defect repair or tissue engineering.

[0020] Compared with the prior art, the present invention is beneficial in that:

[0021] (1) The present invention uses magnetron sputtering technology to prepare an antibacterial film modified with a hydroxyapatite and silver composite coating. The antibacterial film modified with a hydroxyapatite and silver composite coating 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 antibacterial film modified with a hydroxyapatite and silver composite coating 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.

[0022] (2) The present invention effectively utilizes silver and hydroxyapatite to modify the PVDF bone electroactive film through magnetron sputtering technology. The surface of the prepared hydroxyapatite and silver composite coating modified antibacterial film has a silver content of 19.64wt% and a fluorine content of 49.18wt%. The hydroxyapatite and silver composite coating modified antibacterial film can effectively stimulate macrophages to express 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, thereby exerting excellent antibacterial activity; thereby solving the problem of poor antibacterial performance of PVDF bone electroactive film in the prior art.

[0023] (3) The antibacterial film modified by hydroxyapatite and silver composite coating prepared by the present invention can promote osteoblasts to express RUNX family transcription factor 2 (RUNX2), indicating that it has good osteogenic induction activity, solving the problem that the PVDF bone electroactive film in the prior art has poor effect in bone bonding and bone repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A schematic diagram of the preparation method of the antibacterial film modified by the hydroxyapatite and silver composite coating of the present invention;

[0025] Figure 2 The overall image of the antibacterial film modified by the hydroxyapatite and silver composite coating prepared in Example 1 under a scanning electron microscope;

[0026] Figure 3 The energy spectrum of the antibacterial film modified by the hydroxyapatite and silver composite coating prepared in Example 1;

[0027] 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;

[0028] Figure 5The relative mRNA expression of IL-1β after culturing macrophages RAW264.7 with the films prepared in Example 1 and Comparative Example 1;

[0029] 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;

[0030] 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;

[0031] 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;

[0032] 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;

[0033] 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;

[0034] 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;

[0035] 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;

[0036] 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

[0037] 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.

[0038] The present invention provides a method for modifying an antibacterial film by using a composite coating of hydroxyapatite and silver, comprising the following steps:

[0039] 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;

[0040] 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;

[0041] S3, using silver and hydroxyapatite crystals as sputtering sources, in an inert gas atmosphere, sputtering and depositing silver and hydroxyapatite on the PVDF bone electroactive square film to obtain a hydroxyapatite and silver composite coating modified antibacterial film. The schematic diagram of the method for preparing a hydroxyapatite and silver composite coating modified antibacterial film using magnetron sputtering technology of the present invention is as follows Figure 1 shown.

[0042] 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.

[0043] In some examples, in step S3, the gas pressure of the magnetron sputtering is 0.5-5 Pa.

[0044] In some examples, in step S3, the distance between the sputtering source and the square film is 4-10 cm.

[0045] In some examples, in step S3, the sputtering power is 0-100 W.

[0046] In some examples, in step S3, the sputtering temperature is 25-300°C.

[0047] In some examples, in step S3, the inert gas is argon gas, and the flow rate of the argon gas is 5-80 sccm.

[0048] In some examples, in step S3, the sputtering time is 10-20 min.

[0049] Example 1

[0050] A hydroxyapatite and silver composite coating modified antibacterial film, the preparation steps of which are as follows:

[0051] S1. Cut a 28 μm thick PVDF bone electroactive membrane (PolyK, USA) into a square film with an area of ​​2 cm x 2 cm. Ultrasonic clean the film with anhydrous ethanol for 15 min, blow dry with nitrogen, and store it in a vacuum for later use.

[0052] 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;

[0053] S3. Install silver and hydroxyapatite crystals 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 and hydroxyapatite 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 300 °C, the argon flow rate is 5 sccm, and the duration is 20 min to obtain an antibacterial film modified by hydroxyapatite and silver composite coating.

[0054] The antibacterial film modified by the hydroxyapatite and silver composite coating 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 hydroxyapatite and silver composite coating modified antibacterial 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, the surface of the antibacterial film modified by the hydroxyapatite and silver composite coating generated stable silver and hydroxyapatite, among which the silver content was 19.64wt% and the fluorine content was 49.18wt%.

[0055] Table 1: Spectrum scanning specific component results

[0056]

[0057] Example 2

[0058] A hydroxyapatite and silver composite coating modified antibacterial film, the preparation steps of which are as follows:

[0059] S1. Cut a 28 μm thick PVDF bone electroactive membrane (PolyK, USA) into a square film with an area of ​​2 cm x 2 cm. Ultrasonic clean the square film with anhydrous ethanol for 15 min, blow dry with nitrogen, and store it in a vacuum for later use.

[0060] 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;

[0061] S3. Install silver and hydroxyapatite crystals 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 0.5 Pa, and deposit silver and hydroxyapatite 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 300 °C, the argon flow rate is 5 sccm, and the duration is 30 min to obtain an antibacterial film modified by hydroxyapatite and silver composite coating.

[0062] Example 3

[0063] A hydroxyapatite and silver composite coating modified antibacterial film, the preparation steps of which are as follows:

[0064] S1. Cut a 28 μm thick PVDF bone electroactive membrane (PolyK, USA) into a square film with an area of ​​2 cm x 2 cm. Ultrasonic clean the square film with anhydrous ethanol for 15 min, blow dry with nitrogen and store it in a vacuum.

[0065] 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;

[0066] S3. Install silver and hydroxyapatite crystals 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 0.5 Pa. In an argon atmosphere, silver and hydroxyapatite are deposited on a square film, wherein the sputtering distance is 5 cm, the sputtering power is maintained at 50 W, the sputtering temperature is 200 °C, the argon flow rate is 80 sccm, and the duration is 20 min, thereby obtaining an antibacterial film modified by a hydroxyapatite and silver composite coating.

[0067] Comparative Example 1

[0068] A PVDF bone electroactive film, the preparation steps of which are as follows:

[0069] A 28 μm thick PVDF bone electroactive membrane (PolyK, USA) was cut into square films with an area of ​​2 cm x 2 cm. The film was ultrasonically cleaned with anhydrous ethanol for 15 min and dried with nitrogen to obtain the PVDF bone electroactive membrane.

[0070] Comparative Example 2

[0071] A PVDF bone electroactive film, the preparation steps of which are as follows:

[0072] S1. Cut a 28 μm thick PVDF bone electroactive membrane (PolyK, USA) into a square film with an area of ​​2 cm x 2 cm. Ultrasonic clean the film with anhydrous ethanol for 15 min, blow dry with nitrogen, and store it in a vacuum for later use.

[0073] 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;

[0074] S3. Install hydroxyapatite crystals on 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 hydroxyapatite on the square film under argon atmosphere; wherein, the sputtering distance is 10 cm, the sputtering power is maintained at 100 W, the sputtering temperature is 300 °C, the argon flow rate is 5 sccm, and the duration is 20 min, to obtain a PVDF bone electroactive film modified with hydroxyapatite crystals.

[0075] Comparative Example 3

[0076] A PVDF bone electroactive film, the preparation steps of which are as follows:

[0077] S1. Cut a 28 μm thick PVDF bone electroactive membrane (PolyK, USA) into a square film with an area of ​​2 cm x 2 cm. Ultrasonic clean the film with anhydrous ethanol for 15 min, blow dry with nitrogen, and store it in a vacuum for later use.

[0078] 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;

[0079] S3. Install 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 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 300 °C, the argon flow rate is 5 sccm, and the duration is 20 min to obtain a silver-modified PVDF bone electroactive film.

[0080] 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 ImageJ 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 macrophages RAW264.7 were cultured with the films prepared in Example 1 and Comparative Example 1; 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 antibacterial film modified by hydroxyapatite and silver composite coating can promote 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 and enhance the anti-inflammatory activity of macrophages and make them have stronger antibacterial properties.

[0081] 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). ImageJ 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 antibacterial film modified by hydroxyapatite and silver composite coating can upregulate the expression of RUNX2 transcription factor in osteoblasts, indicating that the film has better osteogenic activity.

[0082] Furthermore, the inventors have experimentally verified that the antibacterial films modified by the hydroxyapatite and silver composite coating prepared in Examples 2-3 have excellent biocompatibility, antibacterial properties and osteoinduction activity.

[0083] In summary, the present invention uses magnetron sputtering technology to modify the PVDF bone electroactive film using silver and hydroxyapatite, and the prepared antibacterial film modified by hydroxyapatite and silver composite coating has better antibacterial performance 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 modified silver-hydroxyapatite doped PVDF bone electroactive 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.

[0084] 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 modifying an antibacterial film by a hydroxyapatite and silver composite coating, 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 silver and hydroxyapatite crystals as sputtering sources, in an inert gas atmosphere, silver and hydroxyapatite are sputtered and deposited on a square film to obtain a hydroxyapatite and silver composite coating modified antibacterial film.

2. The method for modifying an antibacterial film by using a hydroxyapatite and silver composite coating 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 modifying an antibacterial film by using a hydroxyapatite and silver composite coating according to claim 1, characterized in that: The gas pressure of the magnetron sputtering is 0.5-5 Pa.

4. The method for modifying an antibacterial film by using a hydroxyapatite and silver composite coating 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 modifying an antibacterial film by using a hydroxyapatite and silver composite coating according to claim 1, characterized in that: The sputtering power is 50-100W.

6. The method for modifying an antibacterial film by using a hydroxyapatite and silver composite coating according to claim 1, characterized in that: The sputtering temperature is 25-300°C.

7. The method for modifying an antibacterial film by using a hydroxyapatite and silver composite coating 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 modifying an antibacterial film by using a hydroxyapatite and silver composite coating according to claim 1, characterized in that: The sputtering time is 10-20 min.

9. An antibacterial film modified by a hydroxyapatite and silver composite coating prepared by the method according to any one of claims 1 to 8.

10. Use of the antibacterial film modified by the hydroxyapatite and silver composite coating as claimed in claim 9 in bone defect repair or tissue engineering.

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