| BackgroundDue to the skin integrity has been damaged, open wounds lose the ability to fend off bacterial invaders. The necrotic tissue and exudate that full of proteins in wound is a good medium for bacteria breeding. Open wounds are easy to be infected, which may even cause systemic infection with the action of many factors. All of that would result in delay of healing period. Recently, the researchers found that the wound may recover faster in the moist environment than that in dry one. The effect on surrounding skin, such as maceration, will reduce the host defensive barriers against microbial invasion provided by the thick skin in the region. On the other hand, in dry environment, cellular activities will be inhibited and form an eschar, and more tissue necrosis may occur at the wound bed. According to this consensus, it is of great significance to develop a high absorptive dressing with the ability to keep the wound wet, for the treatment of surgical wound infection and chronic ulcerative wounds.With the development of modern medicine and the wide application of biological materials, an ideal dressing should possess the following biological performance:① that can absorb exudate of excess,meanwhile maintain a certain humidity on contact with the wound surface;② has excellant biocompatibility;③ is easy to remove that will not cause injury wound again;④ has certain antibacterial activity;⑤ the production and degradation process will not cause environmental pollution.Alginate fibers which are obtained from alginate salt, contain a variety of amino acids, and has good biological compatibility. Alginate fibers can get crosslinking and formed hydrogel that has open mesh lattice under the condition of the presence of calcium ion.The hydrophilic hydrogel, in which the cells may exchange nutrients of diffusion and metabolism of material, provides moist wound healing environment Calcium alginate fibers, made with alginate salt, is non-toxic, high absorptive, good biocompatibility, easy to remove, good biodegradability, hemostatic performance that can promote wound healing.The superior properties makes it widely used for biological implants, artificial tooth filled, biological filler and etc.It is a long history for silver to be used as antibacterial agent. Silver ions, soluble silver compounds, colloidal silver with large surface area and silver nanoparticles have strong antibacterial capacity to kill bacteria, fungi. Based on nanotechnology, silver nanoparticles is a new type of antibacterial products. Because of its quantum effect, small size effect and great specific surface area, silver nanoparticles is stronger than traditional inorganic antibacterial agent in the antibacterial effect.In addition, nanosize silver particles has a strong permeability that can penetrate into the subcutaneous tissue.After contacting with the incision, A dressing loaded with silver nanoparticles wound release nano silver persistently, that kills pathogens on the incision and suture quickly and efficiently. Now, antibacterial products that on the basis of the nano silver, has been widely used in clinic.In this study,we prepared calcium alginate-carboxymethyl cellulose complex by wet spinning, and the AgNPs-Ca were made by dipping and enrichment. The development of the dressing are of great significance for the prevention and treatment of surgical wound infection, promoting healing of chronic wounds.ObjectiveTo develop a novel antibacterial dressing, silver nanoparticle-calcium alginate, whose physics characterization wound be tested. After that, its cytotoxicity, antibacterial properties and biocompatibility wound also be fully evaluated.Then, according to the result, the optimal amount of silver antibacterial dressing wound be shifed as high quality dressing for its further application in the prevention of surgical wound infection, promotion in wound healing.Methods1 The preparation of AgNPs-CaIn this project, AgNPs-Ca was prepared by wet spinning and dipping enrichment methods, more detail as follows:wet spinning:the sodium alginate and carboxymethyl cellulose sodium were dissolved in the solution with the ratio of 8.5:1.5, then stirring in high speed, and form the viscous solution. After deaeration filtered, the solution was squeezed out through the jet hole to the coagulation bath that full of calcium ion and formed solid calcium alginate carboxymethyl fiber filament. After been stretched, washed, dehydrated, dried, crimped, the filament fiber was combed into a net and made a nonwoven fabric.Enrichment and impregnation method:2000 mg/L nano silver solution was diluted with deionized water into different concentrations solution, adding amount of calcium alginate dressings carboxy methyl cellulose and shock in 37℃ for 30 minutes. After dehydration, washed with ethanol, stoved in temperature gradient, and -20℃ for the night, the mass fraction of 0.5%,1%,2%,4% of the calcium alginate silver nanoparticles carboxymethyl cellulose compound was prepared, and silver nanoparticles-calcium alginate antibacterial dressing for short, or AgNPs-Ca.2 The physical characterization of AgNPs-Ca2.1 Ultraviolet-visible spectrophotometer detect absorption spectral characteristics of the dressingsAfter Soaked in deionized water, sample was oscillated for 30 min, and filtered calcium alginate fibers out. The filtered solution was tested with ultraviolet-visible spectrophotometer(NanoDrop 2000),and 0.5 nm scanning interval, finally to obtain the particle distribution curve.2.2 Optical microscope test of dressing samplesSmall sample was tored into a dispersion fiber, then resin and seal. After drying, dressing slides can be preserved for a long-term.After that, the glass was observed and taken pictures with a Olympus optical microscope.2.3 SEM analysis of dressing samplesThe AgNPs-Ca were cut into the size of 8mm X 8mm,5 mm thick sample, and was put into 60℃ dryers for 2 hours,dry thoroughly. Then it was double fixed specimens with glutaraldehyde and osmic acid after high pressure sterilization, then cover platinum on the sample surface by metal coating method,which were put under scanning electron microscope to observe the microstructure of morphology and distributions of fiber and silver nanoparticles.3 The in vitro cytotoxicity test of AgNPs-Ca2ml extract liquid of each group, including gauze, calcium alginate dressings, 0.5%-AgNPs-Ca,1%-AgNPs-Ca,2%-AgNPs-Ca,4%-AgNPs-Ca, Ag+-Ca group, were injected in 35 mm cell cultures respectively, with 2 X 104/ml LO2 cells suspension 2 ml in it.Then they were placed in the incubator for 24 h.Afer that inverted microscope was used to observe the morphology and growth of cells.4 Study on antibacterial properties AgNPs antimicrobial dressings in vitro4.1 Inhibition zoneThe colony of Staphylococcus aureus, Escherichia.coli, Pseudomonas aeruginosa, Candida albicans were selected to made the bacteria suspension with the concentration of 1×106 cfu/ml. Bacteria suspension was smeared on culture plate along by a cotton swab in three directions evenly, and daub on the edge of the culture plate in the end.After covered and dried for 5 min, the culture plates are divided into five parts, which received 10 mm diameter disk of the calcium alginate dressings, 0.5%-AgNPs-Ca,1%-AgNPs-Ca,2%-AgNPs-Ca,4%-AgNPs-Ca in turn clockwise direction. Finally they were put into 37℃ CO2 incubator for 18-20 h and photographed. Image J software was used to survey and map the bacteriostatic circle diameter.4.2 Oscillation bacteriostatic method5 ml bacteria liquid, whose concentration was 3×104cfu/ml, was added into centrifuge tube.The sample of gauze, calcium alginate dressings,0.5%-AgNPs-Ca, 1%-AgNPs-Ca,2%-AgNPs-Ca,4%-AgNPs-Ca, Ag+-Ca were cut into 10 x 10mm square plate, and ultraviolet disinfection for 2 h.Then 2 of each block were placed in the centrifuge tube.All of the tubes were put in a constant temperature shaker under the condition of 25℃ and 200 r/min,100 ul of bacteria liquid were taken out before and 1 h,2 h after oscillation and coated plates and cultivation for 18 h before counting, calculate the bacteriostatic rate of 1h,2h, and appropriate concentration was selected to the next phase of the experiment.4.3 The antimicrobial durability test within 48h5 ml bacteria liquid, in concentration of 3x104cfu/ml, was added into centrifuge tube.The sample of gauze, calcium alginate dressings, 1%-AgNPs-Ca,Ag+-Ca were cut into 10x10mm square plate, and ultraviolet disinfection for 2 h.Then 2 of each block were placed in the centrifuge tube.All of the tubes were put in a constant temperature shaker under the condition of 25℃ and 200 r/min for 2h, and then kept in 37℃ bacteriological incubator for 48h.100 ul of bacteria liquid were taken out before and 2h,4h,6h,24h,48h after oscillation and coated plates and cultivation for 18 h before counting.5 Study on the promotion of wound healing test of AgNPs-Ca24 wistar rats were divided into four groups. After anesthetized and skin preparation, a full-thickness skin area of 180mm2 was cut on each rat,s back. The rats with skin defects were treated with 1%-AgNPs-Ca, Ag+-Ca,calcium alginate dressings and gauze, respectively and change dressing regularly.The wound healing condition were recorded in the day 0,4,8 and 10 after operation, and the wound area measurements using ImageJ software to calculate the rate of wound healing at each time point.The rats from four groups were randomly selected in day 4,8 after operation, whose skin wound were biopsy. After using paraformaldehyde fixed paraffin-embedded and HE staining, the skin organization by observed with microscopy.6 Study on the biocompatibility test of AgNPs-CaAccording to GB/T 16886-2008 and ISO-2009 standard requirements, 10993.12,we detected the biocompatibility of 1%-AgNPs-Ca with hemolysis test,acute systemic toxicity test,in order to evaluate its biocompatibility comprehensively.6.1 Hemolysis test:5 ml sample extract subjects (1%-AgNPs-Ca),5 ml saline (negative control group),5 ml deionized water (positive control group), respectively. Observe whether there is significant hemolysis with naked eye after added 0.1ml fresh anticoagulant dilution rabbit blood into solution prepared separately.The absorbance of supernatant fluid were measured with a ELIASA,and evaluate the in vitro blood compatibility of materials.6.2 Acute systemic toxicity test:The extracts of 1%-AgNPs-Ca antibacterial dressing were injected into the abdomen of test group mice according to a dose of lml/20g standard on sterile procedure.The negative control group injected physiological saline, positive control group injected phenol as the positive control group. Observe and record the mice,s eating and drinking, movement, toxic effects, weight, and death for 7 days after injection.Results1 The physical characterization of AgNPs-CaUltraviolet-visible spectrophotometer showed that the absorption peak of AgNPs-Ca was located at 416 nm, and its distribution range was between 350-550 nm. The ultraviolet spectrum of calcium alginate dressing did not show any special absorption peak.The scanning electron microscope (SEM) of AgNPs-Ca showed that the size of calcium alginate fiber was uniform and 10-15μm in diameter,and the silver nanoparticles adhering to fiber surface and the gap were spherical, particle size were between 80-100 nm.2 The in vitro cytotoxicity test of AgNPs-CaIt is significant differences between different experimental group of cell growth rate (F= 305.04, P< 0.001). After 24 h culture, the cell relative growth rate (RGR) of calcium alginate dressings group is 99.66%, and the cell toxicity was grade 0. The RGR of LO2 cells of 0.5%-AgNPs-Ca group and 1%-AgNPs-Ca were 94.0% and 92.8%,respectively, and both of the cell toxicity were grade I, which indicated that they were no cytotoxicity. There was no significant difference between the two groups. The RGR of LO2 cells of 2%-AgNPs-Ca group was 69.4%, and the cell toxicity was grade II, showing that it was mild cell toxicity. The RGR of LO2 cells of 4%-AgNPs-Ca group and Ag+-Ca were 27.2% and 23%, respectively.Either of their cell toxicity was grade III or IV, and both of them were moderate cytotoxicity.3 Study on antibacterial properties AgNPs antimicrobial dressings in vitro3.1 Inhibition zoneThe inhibition zone could not be observed in the group of calcium alginate dressing in all of the four strains.And the inhibition zone of different groups of AgNPs-Ca vary as the concentration of silver nanoparticles changed, which indicated that the antibacterial capacity of AgNPs was dose-dependent.Treated with Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans,the size of inhibiton zone of 0.5%-AgNPs-Ca is between 11.8～13.6 mm. The size of inhibiton zone of 1%-AgNPs-Ca is between 13.0-14.7 mm. The size of inhibiton zone of 2%-AgNPs-Ca is between 13.5-16.2 mm. The size of inhibiton zone of 4%-AgNPs-Ca is between 15.3-18.0 mm.3.2 Oscillation bacteriostatic methodThe antibacterial rate of different groups varied as treated with Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans in 1h.The antibacterial rate of gauze group was 0%. The antibacterial rate of calcium alginate group was 13.9%,0%,7.4%,9.7%, respectively. The antibacterial rate of 0.5%-AgNPs-Ca group was 58%、26.1%、45.7%、79.3%. The antibacterial rate of 1%-AgNPs-Ca group was 74.6%、48.7%、57.2%、90.4%. The antibacterial rate of 2%-AgNPs-Ca group was 96.8%、60.1%、62.9%、95.4%. The antibacterial rate of 4%-AgNPs-Ca group was 99.7%、88.7%、67.9%、100%. The antibacterial rate of Ag+-Ca group was 50.4%、98.3%、72.0%、37.9%.The antibacterial rate of different groups varied as treated with Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans in 2h.The antibacterial rate of gauze group was all 0%. The antibacterial rate of calcium alginate group was all 0%. The antibacterial rate of 0.5%-AgNPs-Ca group was 90.9%、 87.8%、85.0%、95.6%. The antibacterial rate of 1%-AgNPs-Ca group was 91.2%、 98.0%、89.4%、97.8%. The antibacterial rate of 2%-AgNPs-Ca group was 99.7%、 98.0%、93.8%、99.1%. The antibacterial rate of 4%-AgNPs-Ca group was 100%、 100%、98.5%、100%. The antibacterial rate of Ag+-Ca group was 82.9%、100%、 98.0%、90.4%.Taking the results of cytotoxic test, inhibition zone and oscillation bacteriostatic method into consider,1%-AgNPs-Ca was shifted to be tested in the next experiment.3.3 The antimicrobial durability test within 48hFour strains of bacteria treated with gauze and calcium alginate groups increase significantly in 4 h. And in 24h,the bacteria concentration reached the peak, between 108-109 cfu/ml.There was not change of the concentration in 48 h significantly.The restraint curves of 1%-AgNPs-Ca group were similar in the four bacteria concentration. The bacteria concentration significantly decline in 2 h, and the presence of bacteria were unable to detect in 4h, and maintain 48 h. The restraint curves of Ag+-Ca group is similar to 1%-AgNPs-Ca group, but the inhibition rate reach 100% just in 2 h when treated with E. coli, and the presence of bacteria can’t be detected within the next 48 h.4 Study on the promotion of wound healing test of AgNPs-CaOn day 4, wound healing rate of four groups was no significant difference and the rate was about 20%. On day 8, the wound healing of AgNPs-Ca group that was 70.2%, was higher than Ag+-Ca group, calcium alginate group, gauze groups, and the P values were less than 0.05. The healing rate of Ag+-Ca and calcium alginate group was 50.9%and 55.9% and it was no significant difference between the two groups, the wound healing of gauze groups that was 37.4%, was less than AgNPs-Ca group Ag+-Ca group, calcium alginate group, and the P values were less than 0.05.5 Study on the biocompatibility test of AgNPs-CaHemolysis experiment:1%-AgNPs-Ca extract did not cause hemolysis reaction in vitro.The hemolysis ratio of 1%-AgNPs-Ca antibacterial dressing extracts was 2.79%.Acute systemic toxicity test:There were not abnormal change of eating, breathing, activities in mice on the experimental group after injection within 7days.while body weight increased, and convulsions, unsteady gait,mania and other toxic reactions were not observed, without death.Conclusions1 In this study,we prepared calcium alginate-carboxymethyl cellulose complex by wet spinning, and the AgNPs-Ca were made by dipping and enrichment.It was confirmed that 1%-AgNPs-Ca has excellent antibacterial property, promoting wound healing effect and well biocompatibility in the in vivo and in vitro experiment.2 Tested by ultraviolet-visible spectrophotometry and scanning electron microscopy,it confirmed that nanosize silver particles may released when AgNPs-Ca dissolved in water, and particles have low dispersion.In addition, the fibre diameter of calcium alginate was uniform, and the nanosize silver particles distributed evenly on the fiber surface.3 Cell toxicity tests confirmed that cytotoxicity of AgNPS-Ca was dose-dependent,and 0.5%,1%-AgNPs-Ca remained no cytotoxicity.4 According to the regulations of QB/T 2591-2003, Materials can be referred to as antimicrobial materials when its anti-bacterial rate>90%. It was comformed that AgNPs-Ca have excellent antibacterial properties after tested with inhibition zone and oscillation bacteriostatic method. What’s more,1%-AgNPs-Ca is proved to have antimicrobial durability.5 Animal experiments confirmed that AgNPs-Ca dressings can significantly improve wound healing and reduce the inflammation, reduce secondary damage when change the dressing.6 According to GB/T 16886-2008 and ISO 10993-16886 series of standards for the biomaterial biocompatibility and detection method,1%-AgNPs-Ca does not cause hemolysis, and have no acute systemic toxicity reaction.1%-AgNPs-Ca has good biocompatibility, which was a ideal antimicrobial dressings for further application in the clinical experiment. |
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