Bionic antibacterial nano-composite film as well as preparation method and application thereof

Abstract

The invention discloses a bionic antibacterial nano composite film as well as a preparation method and application thereof. The bionic antibacterial nano composite film comprises an organic matrix, a calcium silicate nanowire and a silver nanowire, the calcium silicate nanowires and the silver nanowires are uniformly dispersed in the organic matrix, and are layered and jointly assembled into a bionic spiral structure. The preparation method of the bionic antibacterial nano composite membrane comprises the following steps: mixing a colloidal solution, rotating the plane of the substrate, spirally assembling a precursor from bottom to top by a one-way brushing and layer-by-layer drying method, putting the precursor into a calcium chloride solution for crosslinking, and the like. The nano-composite membrane disclosed by the invention has relatively strong mechanical properties, good biocompatibility and good osteogenesis-promoting antibacterial ability, and has a good effect of promoting bone tissue regeneration of alveolar bone when being used as a barrier membrane; the preparation method avoids the addition of exogenous growth factors, the required experimental conditions are simple, and the production mode is easy to popularize and large-scale.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, in particular to a bionic antibacterial nanocomposite film, a preparation method and application thereof. Background Art

[0002] Alveolar bone defects caused by tumors, trauma, inflammation, and other reasons can affect the patient's quality of life. Currently, one of the standard treatment procedures for alveolar bone defects is guided bone regeneration technology. This technology places a barrier membrane between the alveolar bone defect and the soft tissue to provide cells with a stable bone formation space, while preventing other cells that may interfere with the healing process from migrating to the defect area, ultimately achieving a predictable bone regeneration effect. Biodegradable collagen membranes are commonly used barrier membrane materials for guided bone regeneration procedures. They have good biocompatibility and certain barrier functions. However, their rapid degradation rate, insufficient mechanical strength, limited regeneration potential, and lack of antibacterial properties limit their clinical regeneration effects.

[0003] Therefore, how to develop an antibacterial barrier membrane with high mechanical strength and good bone regeneration effect is a technical problem that needs to be solved urgently. Summary of the Invention

[0004] Due to the above-mentioned defects in the existing technology, the present invention provides a bionic antibacterial nanocomposite membrane and a preparation method thereof. The membrane adopts an organic matrix mixed with calcium silicate nanowires and silver nanowires to imitate the spiral stacking structure in nature, thereby forming an antibacterial nanocomposite membrane with high mechanical strength and the effect of promoting bone tissue regeneration.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a biomimetic antibacterial nanocomposite membrane comprising an organic matrix, calcium silicate nanowires, and silver nanowires; the calcium silicate nanowires and silver nanowires are uniformly dispersed in the organic matrix and assembled in layers into a biomimetic spiral structure.

[0007] The above technical solution utilizes a bionic spiral structure to co-assemble two nanowires to obtain an antibacterial nanocomposite membrane.

[0008] Furthermore, the organic matrix includes a natural bioactive hydrogel and an auxiliary molding material; the natural bioactive hydrogel includes silk fibroin; and the auxiliary molding material includes sodium alginate. The organic matrix has good biocompatibility with the natural bioactive hydrogel; and the auxiliary molding material includes a bioactive material that, when combined with the inorganic nanowires, exhibits good film-forming properties.

[0009] Furthermore, the mass ratio of the organic matrix to all inorganic substances is 4:1.

[0010] Furthermore, the mass ratio of the silk fibroin to the sodium alginate is 1:1, and the mass ratio of the calcium silicate nanowires to the silver nanowires is 1:1.

[0011] The above material ratio can ensure that the mechanical properties of the membrane meet the application requirements and can form a good membrane.

[0012] In a second aspect, the present invention provides a method for preparing a biomimetic antibacterial nanocomposite film, which is used to prepare the biomimetic antibacterial nanocomposite film as described above, comprising the following steps:

[0013] S11, adding calcium silicate nanowires and silver nanowires to the organic matrix, and mixing them uniformly to obtain a colloidal solution;

[0014] S12, applying the colloidal solution unidirectionally on a plane, and after one layer of colloidal solution dries, applying the next layer unidirectionally; rotating the plane back and forth along the center line so that adjacent layers have a certain angle, and applying the next layer after each rotation; repeating this process multiple times to spirally assemble the precursor from bottom to top;

[0015] S13, placing the spirally assembled precursor into a calcium chloride solution for cross-linking to obtain a biomimetic antibacterial nanocomposite membrane.

[0016] The above preparation method is easy to scale up and has low cost. It can well imitate the natural spiral stacking structure to form an antibacterial nanocomposite film that promotes bone tissue regeneration.

[0017] Furthermore, the angle in S12 is 20°. Setting this angle can mimic the spiral structure of the mantis shrimp's shell. Experimental verification shows that the antibacterial nanocomposite membrane prepared using these experimental parameters exhibits superior mechanical properties and bone regeneration-promoting properties.

[0018] Furthermore, the calcium silicate nanowires are prepared by hydrothermal treatment of calcium nitrate and sodium silicate solutions. The hydrothermally prepared calcium silicate nanowires have a similar length and aspect ratio range to commercially available silver nanowires, which enables the nanowires to have good dispersion properties in organic matrices.

[0019] Furthermore, the organic matrix is a mixture of sodium alginate solution and silk fibroin solution. These two organic matrices have a good dispersion effect on calcium silicate nanowires and silver nanowires.

[0020] Furthermore, the silk fibroin solution is prepared by the following steps:

[0021] S21, degumming the silk in a sodium carbonate solution and drying it thoroughly;

[0022] S22, completely dissolving the degummed product in a lithium bromide solution to obtain a mixed solution;

[0023] S23, placing the mixed solution into a dialysis bag for dialysis to remove impurities, and finally obtaining a silk fibroin solution.

[0024] In the final aspect, the present invention provides an application of a biomimetic antibacterial nanocomposite membrane in promoting alveolar bone tissue regeneration.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] This invention utilizes naturally extracted silk fibroin and sodium alginate solution, adds osteogenesis imperative calcium silicate, and antibacterial silver, mimicking the natural biomimetic spiral structure to form an antibacterial biomimetic nanocomposite membrane for alveolar bone regeneration. This nanocomposite membrane exhibits strong mechanical properties, excellent biocompatibility, and robust osteogenesis and antibacterial properties, effectively promoting alveolar bone regeneration as a barrier membrane.

[0027] The preparation method of the nanocomposite membrane of the present invention avoids adding exogenous growth factors, requires simple experimental conditions, and only involves a stirrer, a heating table, etc. The production method is easy to promote and scale up; the operation method is simple and easy to repeat, and has broad industrialization prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present invention and its features and advantages will become more apparent upon reading the detailed description of non-limiting embodiments made with reference to the following accompanying drawings.

[0029] Figure 1 This is a scanning electron microscope (SEM) image of the inorganic nanowires used in the bionic nanocomposite membrane in an embodiment of the present invention.

[0030] Figure 2 : This is the polarized light and dark change of the cross section of the bionic nanocomposite film in the embodiment of the present invention.

[0031] Figure 3 This is an SEM image of the cross section of the biomimetic nanocomposite membrane in an embodiment of the present invention.

[0032] Figure 4 This is an SEM magnified image and element distribution (EDS) diagram of the cross section of the biomimetic nanocomposite membrane in an embodiment of the present invention.

[0033] Figure 5 This is a test diagram of the tensile mechanical properties of the bionic nanocomposite membrane in an embodiment of the present invention.

[0034] Figure 6 Schematic diagram of live and dead cell staining of the biomimetic nanocomposite membrane in an embodiment of the present invention.

[0035] Figure 7 This is a bacterial plate coating diagram of the blank group and the biomimetic nanocomposite membrane experimental group in the examples of the present invention.

[0036] Figure 8 These are Micro-CT reconstruction images of the blank group, Bio-Gide group, and biomimetic nanocomposite membrane experimental group in the examples of the present invention. DETAILED DESCRIPTION

[0037] The structure of the present invention is further described below with reference to the accompanying drawings and specific embodiments, but is not intended to limit the present invention.

[0038] The reaction devices, compounds, solvents, etc. involved in the following embodiments and examples are all commercially available, and their preparation methods are not described in detail in the present invention.

[0039] The detection instruments and detection reagents involved in the following effect embodiments are all commercially available, and the detection methods adopted are the existing technologies that can be retrieved.

[0040] The present invention provides a biomimetic antibacterial nanocomposite membrane comprising an organic matrix, calcium silicate nanowires, and silver nanowires. The calcium silicate nanowires and silver nanowires are uniformly dispersed in the organic matrix and co-assembled into a biomimetic helical structure. The organic matrix preferably comprises a natural bioactive hydrogel and an auxiliary molding material; the natural bioactive hydrogel comprises silk fibroin; and the auxiliary molding material comprises sodium alginate.

[0041] The preparation method of the bionic antibacterial nanocomposite film comprises the following steps:

[0042] S11, adding calcium silicate nanowires and silver nanowires to the organic matrix, and mixing them uniformly to obtain a colloidal solution;

[0043] S12, applying the colloidal solution unidirectionally on a plane, and after one layer of colloidal solution dries, applying the next layer unidirectionally; rotating the plane back and forth along the center line so that adjacent layers have a certain angle, and applying the next layer after each rotation; repeating this process multiple times to spirally assemble the solution phase from bottom to top;

[0044] S13, placing the spirally assembled solution phase into calcium chloride for cross-linking to obtain a biomimetic antibacterial nanocomposite membrane.

[0045] The technical solutions of the present invention are described in detail below with reference to specific embodiments and comparative examples.

[0046] Example

[0047] The preparation method of the biomimetic antibacterial nanocomposite film of this embodiment comprises the following steps:

[0048] (1) Preparation of calcium silicate nanowires

[0049] 1) Chemical reagents used:

[0050] Calcium nitrate and sodium silicate were used for analysis.

[0051] 2) Experimental instruments used

[0052] Magnetic stirrer, reactor, ultrasonic oscillator.

[0053] Specific steps:

[0054] (1-1) Calcium nitrate and sodium silicate were mixed in a 1:1 molar ratio and placed on a magnetic stirrer for uniform stirring at a speed of 500-1000 rpm. After uniform stirring, the mixed solution was transferred to a reactor for hydrothermal treatment at 200°C.

[0055] (1-2) After cooling naturally to room temperature, the solution was collected and filtered, and the resulting white product was resuspended in deionized water and filtered again three times to wash away residual salts.

[0056] (1-3) Calcium silicate nanowires were obtained after ultrasonic treatment.

[0057] (2) Preparation of silk fibroin solution

[0058] 1) Chemical reagents used:

[0059] Bombyx mori silk for commercial use, sodium carbonate for analysis, and lithium bromide.

[0060] 2) Experimental instruments used

[0061] Magnetic stirrer, dialysis bag.

[0062] Specific steps:

[0063] (2-1) The silk was degummed in a sodium carbonate solution at 100°C and dried at 40°C overnight.

[0064] (2-2) The degummed product was dissolved in a 9.3 M lithium bromide solution, placed on a magnetic stirrer, and stirred at a constant speed for 4 hours to obtain a mixed solution.

[0065] (2-3) The mixed solution was placed in a dialysis bag and dialyzed for 3 days to remove impurities. The resulting silk fibroin solution was stored in a -4°C refrigerator. The molecular weight cutoff of the dialysis bag was 3500 MW.

[0066] (3) Preparation of new biomimetic antibacterial nanocomposite membrane

[0067] 1) Chemical reagents used:

[0068] Silver nanowires for commercial use, sodium alginate for analysis, and calcium chloride.

[0069] 2) Experimental instruments used

[0070] Magnetic stirrer, heating table.

[0071] Specific steps:

[0072] (3-1) Dissolve 2 g of sodium alginate in deionized water and stir on a magnetic stirrer until a homogeneous solution is obtained.

[0073] (3-2) The silk fibroin solution and the sodium alginate solution from step (2) are mixed at a silk fibroin to sodium alginate mass ratio of 1:1, and the calcium silicate nanowires and commercially available silver nanowires from step (1) are added at a mass ratio of 1:1. The mass ratio of total organic matter to total inorganic matter is controlled at 4:1. After the quaternary complex is uniformly mixed, a colloidal solution is obtained.

[0074] (3-3) Apply the colloidal solution in one direction on a flat surface, wait for it to dry on a heating table, and then rotate the surface to keep the angle between adjacent layers at 20°. Repeat this process about 20 times.

[0075] (3-4) The precursor after spiral assembly in this bottom-up manner was placed in a 2% by mass calcium chloride solution for cross-linking for 2 hours, and finally a biomimetic antibacterial nanocomposite membrane was obtained.

[0076] like Figure 1 As shown in Figure 2, the prepared calcium silicate wires and silver wires are both nano-scale inorganic materials. Figure 2 Polarized light experiments show that when the direction of the incident light is rotated multiple times by 45 degrees, the reflected light shows alternating light and dark color changes. Figure 3 The cross-sectional morphology of the nanocomposite film is shown in Figure 2. It can be seen that the cross-sectional morphology of the composite film has a periodic spiral arrangement. The polarization light experiment and cross-sectional SEM photos of the nanocomposite film prove that the biomimetic spiral structure of the nanocomposite film of this embodiment is successfully constructed. Figure 4 ,EDS elemental mapping analysis showed that calcium silicate nanowires and silver nanowires were evenly distributed in the helical structure.

[0077] Tensile mechanics experimental test:

[0078] The prepared biomimetic antibacterial nanocomposite membrane was pre-soaked in water for 2 hours. Then, a tensile test was performed on a universal mechanical machine with a 500N load cell. -1 The membrane was stretched at a constant speed with a loading rate of 0.05 and a gauge length of 5 mm until the membrane broke, and the maximum tensile strength and tensile stress were measured. Figure 5 The results of the tensile test of the prepared nanocomposite film are shown. Its tensile strength is distributed in the range of 10~14 MPa, and the corresponding tensile strain is 100%~120%, indicating that the nanocomposite film has certain mechanical strength and good ductility.

[0079] Cell proliferation assay:

[0080] The Cell Viability Assay Kit uses a dual fluorescence staining method based on calcein and propidium iodide, enabling rapid and convenient detection of live and necrotic cells. After 30 minutes of staining, bright green fluorescence staining of live cells and bright red fluorescence staining of the nuclei of necrotic cells can be observed under a fluorescence microscope.

[0081] After 4 days of co-culture of rat bone marrow mesenchymal stem cells with biomimetic antibacterial nanocomposite membrane, the cell activity and cytotoxicity detection kit showed that ( Figure 6 ), the green-stained cells are evenly distributed on the membrane surface (the area circled by red dashed lines in the figure), so the biomimetic antibacterial nanocomposite membrane has good biocompatibility for bone marrow mesenchymal stem cells.

[0082] Antibacterial experiment:

[0083] The plate coating counting method was used to examine the in vitro antibacterial properties of the biomimetic antibacterial nanocomposite film. The biomimetic antibacterial nanocomposite film was mixed with Staphylococcus aureus suspension (10 6 CFU mL -1 ) and co-cultured in a 24-well plate for 18 hours. After diluting 200,000 times with saline, 20 μL of the diluted suspension was evenly spread on the agar plate. After 12 hours, the plate was photographed and counted by measuring the number of bacterial colonies on the agar plate. Figure 7 As shown in the figure, the blank group was not provided with a nanocomposite membrane, while the experimental group in the figure used the nanocomposite membrane prepared in this example. The results showed that the nanocomposite membrane prepared in this example had good antibacterial properties.

[0084] In vivo animal regeneration experiments:

[0085] The 5mm circular skull defect is a representative standard extreme bone defect model. The results obtained on this model indicate that they can be extended to other bone defect scenarios. Therefore, in this animal experiment, a critical-sized circular skull defect model (5 mm in diameter) was established by drilling the rat skull bilaterally after the rat skull skin was incised. A biomimetic antibacterial nanocomposite membrane and Bio-Gide (a double-layer absorbable collagen membrane made from purified porcine collagen) were placed in the skull defect of the rats, respectively, as the biomimetic antibacterial nanocomposite membrane experimental group and the Bio-Gide group with the blank sample as the control. The samples were collected after 8 weeks. The samples were fixed in paraformaldehyde and then scanned by Micro-CT. The reconstruction results showed ( Figure 8 ):Compared with Bio-Gide, the bionic antibacterial nanocomposite membrane showed a significantly increased bone regeneration area. Therefore, the Micro-CT results showed that the bionic antibacterial nanocomposite membrane had a better ability to promote bone regeneration.

[0086] In summary, the present invention provides a biomimetic antibacterial nanocomposite membrane and its preparation method. This membrane utilizes an organic matrix mixed with calcium silicate nanowires and silver nanowires to mimic the naturally occurring spiral stacking structure, resulting in a mechanically strong antibacterial nanocomposite membrane. This nanocomposite membrane exhibits excellent biocompatibility and osteogenic antibacterial properties.

[0087] Those skilled in the art should understand that they can implement variations by combining the prior art with the above embodiments, which will not be described in detail here. Such variations do not affect the essence of the present invention and will not be described in detail here.

[0088] The above describes the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and the devices and structures that are not described in detail should be understood to be implemented in a common manner in the art; any technician familiar with the art can use the above-mentioned disclosed methods and technical contents to make many possible changes and modifications to the technical solutions of the present invention without departing from the scope of the technical solutions of the present invention, or modify them into equivalent embodiments of equivalent changes, which does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention that do not depart from the content of the technical solutions of the present invention are still within the scope of protection of the technical solutions of the present invention.

Claims

1. A biomimetic antibacterial nanocomposite membrane, characterized by: including organic matrix, calcium silicate nanowires, and silver nanowires; The calcium silicate nanowires and silver nanowires are uniformly dispersed in the organic matrix and are layered and assembled into a bionic spiral structure.

2. The biomimetic antibacterial nanocomposite membrane according to claim 1, characterized in that: The organic matrix includes natural bioactive hydrogel and auxiliary molding materials; the natural bioactive hydrogel includes silk fibroin; and the auxiliary molding material includes sodium alginate.

3. A biomimetic antibacterial nanocomposite membrane according to claim 1 or 2, characterized in that: The mass ratio of the organic matrix to all inorganic substances is 4:

1.

4. The biomimetic antibacterial nanocomposite membrane according to claim 2, characterized in that: The mass ratio of the silk fibroin to sodium alginate is 1:1, and the mass ratio of the calcium silicate nanowire to silver nanowire is 1:

1.

5. A method for preparing a biomimetic antibacterial nanocomposite film, characterized in that: The method for preparing the biomimetic antibacterial nanocomposite film according to any one of claims 1 to 4 comprises the following steps: S11, adding calcium silicate nanowires and silver nanowires to the organic matrix, and mixing them uniformly to obtain a colloidal solution; S12, applying the colloidal solution unidirectionally on a plane, and after one layer of colloidal solution dries, applying the next layer unidirectionally; rotating the plane back and forth along the center line so that adjacent layers have a certain angle, and applying the next layer after each rotation; repeating this process multiple times to spirally assemble the precursor from bottom to top; S13. The helical assembled precursor is placed in a calcium chloride solution for cross-linking to obtain a biomimetic antibacterial nanocomposite membrane.

6. The method for preparing a biomimetic antibacterial nanocomposite film according to claim 5, characterized in that: The included angle in S12 is 20°.

7. The method for preparing a biomimetic antibacterial nanocomposite film according to claim 5, characterized in that: The calcium silicate nanowires are prepared by hydrothermal treatment of calcium nitrate and sodium silicate solutions.

8. The method for preparing a biomimetic antibacterial nanocomposite film according to claim 5, characterized in that: The organic matrix is a mixture of sodium alginate solution and silk fibroin solution.

9. The method for preparing a biomimetic antibacterial nanocomposite film according to claim 8, characterized in that: The silk fibroin solution is prepared by the following steps: S21, degumming the silk in a sodium carbonate solution and drying it thoroughly; S22, completely dissolving the degummed product in a lithium bromide solution to obtain a mixed solution; S23, placing the mixed solution into a dialysis bag for dialysis to remove impurities, and finally obtaining a silk fibroin solution.

10. Application of a biomimetic antibacterial nanocomposite membrane in promoting alveolar bone tissue regeneration.

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