| Background and Objective:Damages such as mechanical trauma,burn or chronic inflammation can lead to skin defects.When.the skin tissue is directly exposed to the environment and accompanied by foreign bodies,the damaged skin tissue is vulnerable to invasion by bacteria.Bacteria can produce toxins,pathogenic enzymes and pathogenic factors on the wound surface,which can lead to delayed wound healing,and even can cause ulceration and necrosis in severe cases.Therefore,preventing and reducing bacterial infection is a key factor in promoting healing.Silk fibroin（SF）,a natural protein fiber obtained from silk,is derived from living organisms.Due to the abilities of good biocompatibility,non-toxic and non-irritating,and with a similar structure to the extracellular matrix（ECM）,SF has been widely used in wound dressings.However,SF per se does not have antibacterial function.Silver（Ag）,as an inorganic antibacterial agent,is often used to enhance the antibacterial performance of dressings.Studies have shown that eukaryotic cells are more resistant to Ag than prokaryotic cells.Therefore,the difference between Ag cytotoxicity and antibacterial effect can be used as a therapeutic window for its antibacterial applications.Nevertheless,the uses of Ag are still controversial at present.Some studies have found that Ag-containing dressings could delay wound healing,while others have indicated that Ag-containing dressings could promote wound healing.This may be because that Ag,as a double-edged sword,not only has antibacterial properties but also produces toxic effects on cells.Therefore,this study aims to develop a two-layered structure composite membrane,including a Ag-rich SF layer（Ag-SF）and a pure SF layer.Through a simplified layer-by-layer technique reduces the cytotoxicity of Ag,at the same time maintains its antibacterial properties,to achieve the effect of balancing the cytotoxicity and the antibacterial properties of Ag-loaded materials.Materials and Methods:1.Preparation and characterization of the SF membrane and Ag-SF/SF membrane:SF membrane and Ag-SF/SF membrane were synthesized by the acid-soluble and alcohol-solid method,and the simplified layer-by-layer technique was used to prepare Ag-SF/SF membranes.The morphology of the SF membrane and Ag-SF/SF membrane was observed under a scanning electron microscope（SEM）.The elements distribution of the Ag-SF/SF membrane was analyzed by energy dispersive X-ray（EDX）.The phase and surface functional group analysis results of the SF membrane and Ag-SF/SF membrane were obtained by X-ray diffraction（XRD）and fourier transform infrared（FTIR）.The tensile properties of the SF membrane and Ag-SF/SF membrane were tested by the universal tester.Atomic absorption spectroscopy（AAS）was used to estimate the concentration of Ag+to evaluate the release curve of Ag-SF/SF membranes.2.Antibacterial test of Ag-SF/SF membranes:Ag-SF/SF membranes were placed on the nutrient agar culture plates which were uniformly coated with the suspension of Staphylococcus aureus（S.aureus）or Escherichia coli（E.coli）.After 20 h cultivation in the incubator at 36℃,the antibacterial effects of Ag-SF/SF membranes were tested by measuring the diameter of the transparent inhibition zones.3.Cell viability on SF membrane and Ag-SF/SF membrane:Human foreskin fibroblasts（HFFs）were seeded onto blank tissue culture plates（TCP）,SF membrane,and Ag-SF/SF membrane.After cultivation for 24 h,the cell viability was detected by live/dead cells staining and cell-counting kit-8（CCK-8）.4.Scratch wound healing test on SF membrane and Ag-SF/SF membrane:HFFs were inoculated on SF membrane and the SF side of Ag0.12-SF/SF 1:3 membrane,with HFFs on the TCP as the control.After the confluence,the monolayer HFFs were scratched by the 10 μL sterile pipette tip.The wound healing photographs were taken at 0 and 24 h after the scratch.5.The gene expression levels of HFFs on SF membrane and Ag-SF/SF membrane:HFFs were inoculated on SF membrane and the SF side of Ag0.12-SF/SF1:3 membrane,with HFFs on the TCP as the control.After cultivated for 7 d,the quantitative real-time polymerase chain reaction（qRT-PCR）was used to detect the expression levels of collagen Ⅰ（Col Ⅰ）and transforming growth factor-β（TGF-β）.6.The healing effect of Ag-SF/SF membrane on infected wounds of skin:The experiment involved twelve 8-week-old Wistar male rats.All wounds were randomly divided into 3 groups,namely sham control,SF group,and Ag0.12-SF/SF1:3 group.Four circular wounds of 8-mm-diameter were punched on both sides of the rat back skin by the disposable biopsy punches.After the infected wounds were established by injecting bacterial suspension to the wound surfaces,the wounds were covered with SF membrane or Ag0.12-SF/SF1:3 membrane.Photographs of wound healing were taken at 0,4,7,11,and 14 days after the operation.After calibrating the scale of the wound healing photographs,the wound areas were measured to calculate the reduction rates of the wounds.At day 4 and 14,the rats were euthanized with an overdose of anesthetics and the skin samples were collected.The samples were prepared into paraffin sections and stained with Masson’s trichrome.The new collagen formation in the wound areas was measured and calculated.Results:1.SF membrane and Ag-SF/SF membrane were successfully prepared.SEM results showed that the SF membrane and Ag-SF/SF membrane both had a smooth surface.The EDX result confirmed that the particles on the Ag-SF side of the Ag-SF/SF membrane were AgCl.The XRD patterns showed that both SF membrane and Ag-SF/SF membrane had a typical diffraction peak of silk II,meanwhile,the Ag-SF side of the Ag-SF/SF membrane showed several typical diffraction peaks of AgCl.FTIR analysis revealed that the SF membrane and Ag-SF/SF membrane showed the vibrations of amide Ⅱ and amide Ⅰ at 1517 cm-1 and 1623 cm-1.SF membrane and Ag-SF/SF membrane had similar Young’s modulus and tensile strength.The release concentrations of Ag+were detected by AAS.The results showed that the early release of Ag+was relatively fast,and the subsequent release was relatively slow.2.The zone of inhibition（ZOI）test results showed that the antibacterial effect of Ag-SF/SF membranes rose with the increase of Ag content,however,the antibacterial effect of Ag0.12-SF/SF membranes was equivalent to that of Ag0.24-SF/SF membranes.At the same time,the SF side and Ag-SF side of the same Ag-SF/SF membrane showed comparable antibacterial efficacy.Further comparison of the antibacterial property of membranes with different layer thickness ratios showed that the antibacterial property of Ag-SF/SF membranes decreased gradually with the increase of SF layer thickness.In addition,under the condition of the same Ag content,regardless of the double-layer or single-layer design,the antibacterial effects of membranes were similar with the same thickness.3.Live/dead cell staining and CCK-8 assay results showed that with the increase of Ag content,the cytotoxicity on both sides of the Ag-SF/SF membranes increased correspondingly.However,the cell viability on the SF side of the Ag0.12-SF/SF group was higher than that of the Ag-SF side.Further the effects of materials with different layer thickness ratios on cell viability were explored.The results showed that under the condition of the same Ag content,compared with the uniformly distributed single-layer membranes in the same thickness and the Ag-SF side of the Ag0.12-SF/SF membranes,the SF side of the Ag0.12-SF/SF membranes showed better cytocompatibility.4.The scratch test results showed that after scratching for 24 h,both the SF membrane and the SF side of Ag0.12-SF/SF1:3 membrane showed similar in vitro wound closure to the TCP group.5.The qRT-PCR results showed that after cultivation for 7 d,the expression levels of Col Ⅰ and TGF-β on the SF membrane and the SF side of Ag0.12-SF/SF1:3 membrane were higher than those of the TCP control group.6.Animal experiments showed that all wounds had a healing rate of more than 90%at 14 d,but the healing rate of the Ag0.12-SF/SF1:3 group was significantly faster than that of the sham group and SF group.Masson staining showed that collagen formation in the Ag0.12-SF/SF1:3 group was significantly higher than that in the sham group and SF group at day 4 and 14.Conclusion:In this study,Ag-SF/SF membrane was successfully prepared.Both sides of the Ag-SF/SF membrane exerted antibacterial effects and showed low cytotoxicity on the SF side.Meanwhile,the membrane effectively promoted wound healing.Therefore,this study provides a new strategy to reduce the cytotoxicity of the material by achieving unilateral distribution of Ag using a simplified layer-by-layer technique. |
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