Method for modifying antibacterial film by hydroxyapatite and silver composite coating and application thereof

Abstract

The application provides a method for modifying an antibacterial film with a hydroxyapatite and silver composite coating and application, and belongs to the technical field of material modification. The application takes silver single element and hydroxyapatite crystals as sputtering sources, and prepares a hydroxyapatite and silver composite coating on the surface of a PVDF bone electroactive square film through a magnetron sputtering technology in an inert gas atmosphere to obtain an antibacterial film modified with the hydroxyapatite and silver composite coating. The antibacterial film modified with the hydroxyapatite and silver composite coating has superior biocompatibility, antibacterial performance and osteogenesis induction activity, and effectively solves the problems of poor bone integration, bone healing speed and effectiveness of the PVDF bone electroactive film in the prior art. The antibacterial film modified with the hydroxyapatite and silver composite coating is suitable for tissue engineering and bone defect repair, and expands the application scenarios of the PVDF bone electroactive film in the oral cavity and other parts.

Description

Technical Field

[0001] This invention relates to the field of material modification technology, specifically to a method and application of modifying an antibacterial film with a hydroxyapatite and silver composite coating. Background Technology

[0002] Bone tissue repair and regeneration involve a complex process requiring various 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 a crucial role in bone maturation, remodeling, and reconstruction. Using electroactive materials to fabricate bone repair scaffolds can mimic the physiological electrical microenvironment of bone tissue, thereby promoting bone regeneration and remodeling.

[0003] PVDF (polyvinylidene fluoride polymer) osteoactive films possess excellent piezoelectric properties and a large specific surface area, effectively mimicking the collagen fiber structure in the natural extracellular matrix. They instantaneously generate surface charge upon undergoing minute 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 mechanotransmission, thereby inducing osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Therefore, PVDF osteoactive films have wide applications in bone tissue engineering, bone repair, biosensors, and drug delivery.

[0004] Despite the great potential shown by PVDF osteoactive membranes, several challenges and problems remain in practical applications, such as poor electrical signal stability, poor regulation of bone regeneration, and slow bone healing. Therefore, designing an osteoactive membrane with good biocompatibility, antibacterial activity, stability, and the ability to regulate bone regeneration is of significant clinical importance. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method and application for modifying an antibacterial film with a hydroxyapatite-silver composite coating. This invention utilizes magnetron sputtering technology to modify the antibacterial film with a hydroxyapatite-silver composite coating. The modified antibacterial film exhibits superior biocompatibility, antibacterial properties, and osteogenic induction activity, effectively solving the problems of poor bone integration, slow bone healing, and limited effectiveness of existing PVDF osteoactive films.

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

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

[0008] S1. Cut the PVDF bone electroactive membrane into square films of appropriate size, clean and dry the square films, and store them in a vacuum for later use.

[0009] S2. Adhere conductive adhesive to the glass slide, attach the square film processed in step S1 onto the conductive adhesive, and invert the glass slide and place it into the magnetron sputtering chamber.

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

[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 thin 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, and the flow rate of the argon is 5-80 sccm.

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

[0018] Secondly, the present invention provides a hydroxyapatite-silver composite coating modified antibacterial film prepared by the method described above.

[0019] Thirdly, the present invention provides the application 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 advantages of the present invention are:

[0021] (1) The present invention uses magnetron sputtering technology to prepare a hydroxyapatite and silver composite coating modified antibacterial film. The hydroxyapatite and silver composite coating modified antibacterial film has excellent biocompatibility, antibacterial properties and osteogenic induction activity, which effectively solves the problems of poor bone integration, bone healing speed and effectiveness of the existing PVDF osteoactive film. The hydroxyapatite and silver composite coating modified antibacterial film is suitable for tissue engineering and bone defect repair, which expands the application scenarios of PVDF osteoactive film in the oral cavity and other parts.

[0022] (2) This invention effectively utilizes silver and hydroxyapatite to modify PVDF bone electroactive film using magnetron sputtering technology. The surface of the hydroxyapatite and silver composite coating modified antibacterial film has a silver content of 19.64 wt% and a fluorine content of 49.18 wt%. 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 exhibiting excellent antibacterial activity; thus 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 in this invention can promote the expression of RUNX family transcription factor 2 (RUNX2) in osteoblasts, indicating that it has good osteogenic induction activity, which solves the problem that the PVDF osteoactive film in the prior art has poor effect on bone integration and bone repair. Attached Figure Description

[0024] Figure 1 This is 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 is shown.

[0027] Figure 4 The relative mRNA expression of CD86 in RAW264.7 macrophages after culturing the films prepared in Example 1 and Comparative Example 1 are shown.

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

[0029] Figure 6 The relative mRNA expression of IRF3 in RAW264.7 macrophages after culturing the films prepared in Example 1 and Comparative Example 1 are shown.

[0030] Figure 7 The relative mRNA expression of TRAF6 in RAW264.7 macrophages after culturing the films prepared in Example 1 and Comparative Example 1 are shown.

[0031] Figure 8 The relative mRNA expression of NF-κB in RAW264.7 macrophages after culturing the films prepared in Example 1 and Comparative Example 1, respectively;

[0032] Figure 9 The graph shows the relative mRNA expression of IL-6 in RAW264.7 macrophages after culturing the films prepared in Example 1, Comparative Example 2, and Comparative Example 3.

[0033] Figure 10 The relative mRNA expression of TRAF6 in RAW264.7 macrophages after culturing the films prepared in Example 1, Comparative Example 2, and Comparative Example 3 are shown in the diagram.

[0034] Figure 11 The graphs show the relative mRNA expression of NF-κB in RAW264.7 macrophages after culturing the films prepared in Example 1, Comparative Example 2, and Comparative Example 3.

[0035] Figure 12 The relative mRNA expression of MYD88 in RAW264.7 macrophages after culturing the films prepared in Example 1, Comparative Example 2, and Comparative Example 3 are shown in the diagram.

[0036] Figure 13 The graphs show the relative mRNA expression of RUNX2 in osteoblasts mt-3t3 after the films prepared in Example 1 and Comparative Example 1 were cultured. Detailed Implementation

[0037] The technical solution of the present invention will be clearly and completely described below. Obviously, 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.

[0038] This invention provides a method for modifying an antibacterial film with a hydroxyapatite-silver composite coating, comprising the following steps:

[0039] S1. Cut the PVDF bone electroactive membrane into square films of appropriate size, clean and dry the square films, and store them in a vacuum for later use.

[0040] S2. Adhere conductive adhesive to the glass slide, attach the square film processed in step S1 onto the conductive adhesive, and invert the glass slide and place it into the magnetron sputtering chamber.

[0041] S3. Using elemental silver and hydroxyapatite crystals as sputtering sources, silver and hydroxyapatite are sputtered and deposited onto a PVDF bone electroactive square film under an inert gas atmosphere to obtain a hydroxyapatite-silver composite coating modified antibacterial film. A schematic diagram of the method for preparing a hydroxyapatite-silver composite coating modified antibacterial film using magnetron sputtering technology is shown below. Figure 1 As shown.

[0042] In some examples, in step S1, the square film has an area of ​​1 cm x 1 cm to 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, the sputtering power in step S3 is 0-100 W.

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

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

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

[0049] Example 1

[0050] A hydroxyapatite and silver composite coating modified antibacterial film is prepared by the following steps:

[0051] S1. Cut a 28 μm thick PVDF bone electrokinetic membrane (PolyK, USA) into square films with an area of ​​2 cm x 2 cm. Clean the films with anhydrous ethanol by ultrasonication for 15 min, dry them with nitrogen, and store them in a vacuum for later use.

[0052] S2. Adhere conductive adhesive to the glass slide, attach the square film processed in step S1 onto the conductive adhesive, and invert the glass slide and place it into the magnetron sputtering chamber.

[0053] S3. Silver and hydroxyapatite crystals are installed in the DC target area of ​​magnetron sputtering. After the chamber is closed, high-purity argon gas is introduced to maintain the chamber pressure at 5 Pa. Silver and hydroxyapatite are deposited on a square film under the argon atmosphere. The sputtering distance is 10 cm, the sputtering power is maintained at 100 W, the sputtering temperature is 300 ℃, the argon gas flow rate is 5 sccm, and the duration is 20 min. A hydroxyapatite and silver composite coating modified antibacterial film is obtained.

[0054] The antibacterial film modified with the hydroxyapatite-silver composite coating prepared in Example 1 was imaged using a scanning electron microscope at 10,000x magnification. The results are as follows: Figure 2 As shown. By Figure 2 It can be seen that the surface of the modified PVDF bone electroactive film is relatively smooth. Surface elemental analysis of the obtained hydroxyapatite and silver composite coating modified antibacterial film was performed using an energy dispersive spectroscopy (EDS) module, and the results are as follows: Figure 3 As shown in Table 1. From Figure 3 As shown in Table 1, the surface of the antibacterial film modified by the hydroxyapatite and silver composite coating has a stable content of silver and hydroxyapatite, of which the silver content is 19.64 wt% and the fluorine content is 49.18 wt%.

[0055] Table 1: Specific component results of energy dispersive spectroscopy (EDS) scan

[0056]

[0057] Example 2

[0058] A hydroxyapatite and silver composite coating modified antibacterial film is prepared by the following steps:

[0059] S1. Cut a 28 μm thick PVDF bone electrokinetic membrane (PolyK, USA) into square films with an area of ​​2 cm x 2 cm. Clean the square films with anhydrous ethanol by ultrasonication for 15 min, dry them with nitrogen, and store them in a vacuum for later use.

[0060] S2. Adhere conductive adhesive to the glass slide, attach the square film processed in step S1 onto the conductive adhesive, and invert the glass slide and place it into the magnetron sputtering chamber.

[0061] S3. Silver and hydroxyapatite crystals are installed in the DC target area of ​​magnetron sputtering. After the chamber is closed, high-purity argon gas is introduced to maintain the chamber pressure at 0.5 Pa. Silver and hydroxyapatite are deposited on a square film under an argon atmosphere. The sputtering distance is 10 cm, the sputtering power is maintained at 100 W, the sputtering temperature is 300 ℃, the argon gas flow rate is 5 sccm, and the duration is 30 min. A hydroxyapatite and silver composite coating modified antibacterial film is obtained.

[0062] Example 3

[0063] A hydroxyapatite and silver composite coating modified antibacterial film is prepared by the following steps:

[0064] S1. Cut a 28 μm thick PVDF bone electroactive membrane (PolyK, USA) into square films with an area of ​​2 cm x 2 cm. Clean the square films with anhydrous ethanol by ultrasonication for 15 min, dry them with nitrogen, and then store them in a vacuum.

[0065] S2. Adhere conductive adhesive to the glass slide, attach the square film processed in step S1 onto the conductive adhesive, and invert the glass slide and place it into the magnetron sputtering chamber.

[0066] S3. Silver and hydroxyapatite crystals are mounted on the DC target area of ​​magnetron sputtering. After closing the chamber, high-purity argon gas is introduced to maintain the chamber pressure at 0.5 Pa. Silver and hydroxyapatite are deposited on a square film under an argon atmosphere. The sputtering distance is 5 cm, the sputtering power is maintained at 50 W, the sputtering temperature is 200 ℃, the argon gas flow rate is 80 sccm, and the duration is 20 min, resulting in a hydroxyapatite and silver composite coating modified antibacterial film.

[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 films were 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 electrokinetic membrane (PolyK, USA) into square films with an area of ​​2 cm x 2 cm. Clean the films with anhydrous ethanol by ultrasonication for 15 min, dry them with nitrogen, and store them in a vacuum for later use.

[0073] S2. Adhere conductive adhesive to the glass slide, attach the square film processed in step S1 onto the conductive adhesive, and invert the glass slide and place it into the magnetron sputtering chamber.

[0074] S3. Hydroxyapatite crystals are installed in the DC target area of ​​magnetron sputtering. After the chamber is closed, high-purity argon gas is introduced to maintain the pressure in the chamber at 5 Pa. Hydroxyapatite is deposited on a square film under the argon atmosphere. The sputtering distance is 10 cm, the sputtering power is maintained at 100 W, the sputtering temperature is 300 ℃, the argon gas 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 electrokinetic membrane (PolyK, USA) into square films with an area of ​​2 cm x 2 cm. Clean the films with anhydrous ethanol by ultrasonication for 15 min, dry them with nitrogen, and store them in a vacuum for later use.

[0078] S2. Adhere conductive adhesive to the glass slide, attach the square film processed in step S1 onto the conductive adhesive, and invert the glass slide and place it into the magnetron sputtering chamber.

[0079] S3. Silver element was installed in the DC target area of ​​magnetron sputtering. After the chamber was closed, high-purity argon gas was introduced to maintain the gas pressure in the chamber at 5 Pa. Silver was deposited on a square film under the argon atmosphere. The sputtering distance was 10 cm, the sputtering power was maintained at 100 W, the sputtering temperature was 300 ℃, the argon gas flow rate was 5 sccm, and the duration was 20 min to obtain a silver-modified PVDF bone electroactive film.

[0080] The films prepared in Example 1 and Comparative Examples 1-3 were soaked in alcohol and then sterilized by UV irradiation. They were then placed in six-well cell culture plates and seeded with RAW264.7 macrophages at a density of 50%. Two days later, when the cell density reached 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 plotting were performed using ImageJ software. The results are shown below. Figure 4-12 As shown, where, Figure 4-8 The images show the relative mRNA expression of CD86, IL-1β, IRF3, TRAF6, and NF-κB in RAW264.7 macrophages after culturing the films prepared in Example 1 and Comparative Example 1, respectively. Figure 9-12 The graphs show the relative mRNA expression of IL-6, TRAF6, NF-κB, and MYD88 in RAW264.7 macrophages after culturing the films prepared in Examples 1, 2, and 3, respectively. Figure 4-12 It is known that the antibacterial film modified with 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 enhance the anti-inflammatory activity of macrophages and make them have stronger antibacterial properties.

[0081] The films prepared in Example 1 and Comparative Example 1 were soaked in alcohol and then sterilized by UV irradiation. They were then placed in six-well cell culture plates and seeded with 50% confluence of osteoblasts (mt-3t3). Two days later, when the cell density reached 80%, the cells were collected for qPCR experiments to detect the expression of RUNX family transcription factor 2 (RUNX2). Statistical analysis and plotting were performed using ImageJ software. The results are shown below. Figure 13 As shown. By Figure 13 It can be seen that the antibacterial membrane modified with hydroxyapatite and silver composite coating can upregulate the expression of osteoblast RUNX2 transcription factor, indicating that the membrane has better osteogenic activity.

[0082] Furthermore, the inventors have verified through experiments that the antibacterial films modified with hydroxyapatite and silver composite coatings prepared in Examples 2-3 all exhibit superior biocompatibility, antibacterial properties, and osteogenic induction activity.

[0083] In summary, this invention utilizes magnetron sputtering technology to modify PVDF bone electroactive films with silver and hydroxyapatite. The resulting hydroxyapatite-silver composite coating-modified antibacterial film exhibits superior antibacterial properties and osteogenic activity, effectively addressing the shortcomings of existing PVDF bone electroactive films in terms of poor bone integration, bone healing speed, and effectiveness. Furthermore, the modified silver-hydroxyapatite-doped PVDF bone electroactive film is suitable for tissue engineering and bone defect repair, expanding the application scenarios of PVDF bone electroactive films in the oral cavity and other sites.

[0084] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A method for modifying an antibacterial film with a hydroxyapatite and silver composite coating, characterized in that, Includes the following steps: S1. Cut the PVDF bone electroactive membrane into square films of appropriate size, clean and dry the square films, and store them in a vacuum for later use. S2. Adhere conductive adhesive to the glass slide, attach the square film processed in step S1 onto the conductive adhesive, and invert the glass slide and place it into the magnetron sputtering chamber. S3. Using elemental silver and hydroxyapatite crystals as sputtering sources, silver and hydroxyapatite are sputtered and deposited on a square thin film under an inert gas atmosphere to obtain a hydroxyapatite and silver composite coating modified antibacterial film; the sputtering power is 50-100 W and the sputtering time is 10-20 min.

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

4. The method for modifying an antibacterial film with a hydroxyapatite and silver composite coating according to claim 1, characterized in that, The distance between the sputtering source and the square thin film is 4-10 cm.

5. The method for modifying an antibacterial film with a hydroxyapatite and silver composite coating according to claim 1, characterized in that, The sputtering temperature is 25-300 ℃.

6. The method for modifying an antibacterial film with a hydroxyapatite and silver composite coating according to claim 1, characterized in that, The inert gas is argon, and the flow rate of the argon is 5-80 sccm.

7. A hydroxyapatite-silver composite coating modified antibacterial film prepared by the method described in any one of claims 1 to 6.

8. The application of a hydroxyapatite and silver composite coating modified antibacterial film as described in claim 7 in bone defect repair or tissue engineering.

Images