| Background and Purposes:Bacterial bio film (BBF) is an aggregate of microorganisms in which cells adhere to each other on a surface. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance, which is a polymeric conglomeration generally composed of extracellular DNA, proteins, and polysaccharides. The bacteria within the biofilms show an extremely high level of tolerance to antibiotics, as well as components of the host’s own defense system, which are physiologically distinct from the planktonic cells of the same species. So, biofilms can hardly be eradicated by conventional antibiotic therapy and are thought to provide a nidus for recurrent infection. Nowadays, it is generally believed that BBF is the most common cause of persistent infections and chronic inflammatory diseases which are difficult to cure. Since 2004, biofilms have been consistently identified on the mucosal surface of patients with chronic rhino sinusitis (CRS). The presence of biofilms on the patients’ sinonasal mucosa has been associated with more severe disease preoperatively and poorer outcomes following endoscopic sinus surgery. Although surgical resection can remove the biofilms, complete removal is difficult to achieve by surgery alone. This is because the involving range of BBF can not be distinguished by naked eyes. Recent investigations indicate that long-term treatment with low-dose macrolides has some success in BBF-related respiratory tract diseases (including sinusitis). However, this treatment program also has many deficiencies, such as long treatment period, significant side effects, high cost and poor patient compliance. As the role of BBF in the pathogenesis of CRS has been recognized, the focus of research interest has switched to the development of biofilm eradication strategies. Suitable animal model will be conducive to such research. However, the existing animal models are all have serious flaws which limit the conduct of such research. Staphylococcus aureus (S. aureus) is one of the most common pathogens of human diseases, and investigators have recently identified it is also the most common biofilm-forming organism isolated from patients with chronic sinusitis refractory to medical and surgical therapy. Therefore, S. aureus biofilms were believed to play a dominant role in negatively affecting the outcomes after endoscopic sinus surgery (e.g. persistent postoperative symptoms, mucosal inflammation and infections). And some experts believed that future studies evaluating therapeutic intervention of BBF-related sinusitis should specifically target S. aureus biofilms. Silver nanoparticle is well-known for its anti-bacterial and anti-biofilm properties to a spectrum of bacteria and has been used increasingly in clinical practice. But the nano-silver products are still produced mainly by chemical reduction method, which is complex and needs toxic agents as reducing and stabilizing agents. For clinical use, people are more concerned with the green synthetic strategy for the silver nanoparticles by using non-toxic chemicals, environmentally benign solvents, and renewable materials. Studies show silver nanoparticles, polymeric with biological materials, can increase their stability against aggregation and their potential biocompatibility. The most widely used compounds for the stabilization of metal nanoparticles are ligands and polymers, especially for some polymers which have certain affinity for metals and are soluble in suitable solvents. As a natural biological macromolecule, silk fibroin has been used as biomedical materials for long time because of its good biocompatibility. And recently, studies have shown that silk fibroin can reduce some metal ions to form metal nanoparticles. The purpose of this study:1. To prepare a silk fibroin-nano silver (SF-NS) solution which is suitable for clinical use according to the "green chemistry" concept.2. To observe the anti-bacterial and anti-biofilm properties of the resulted SF-NS solution targeting S. aureus, and explore its potential clinical value.3. To make an animal model of S. aureus biofilm-related sinusitis, and further investigate the efficacy of SF-NS solution as topical anti-biofilm agent in vivo.Materials and Methods:The experiment consists of four parts. The first part is preparation and characterization of SF-NS solution. Silk fibroin was used as a bio-template to in situ prepare silver nanoparticles at room temperature under light exposure. To investigate the reducing power and dynamic indicators of silk fibroin and the morphology character of the formed silver nanoparticles, the reaction process and production were detected by fluorescence spectroscopy, UV-visible spectrophotometer (UV-vis), transmission electron microscopy (TEM), high resolution transmission electron microscopy (HRTEM) and etc. The second part is observation of the anti-bacterial and anti-biofilm properties of SF-NS solution targeting S. aureus. The anti-bacterial activity of SF-NS solution was evaluated by determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against biofilm positive clinical strain of S. aureus and control ATCC 29213 in vitro. MIC and MBC testing were performed according to the Clinical and Laboratory Standards Institute (CLSI,2011) guidelines for macro-dilution in broth. Then, the anti-biofilm properties of the SF-NS solution were determined by Congo Red agar method, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The third part is establishment of S. aureus biofilm-related sinusitis animal model. Forty five New Zealand white rabbits (2.5-3.5 kg) were randomly divided into nine groups, including four model groups, four negative control groups and one blank control group, five rabbits in each group. The model rabbits were made by means of gelatin sponge placement and inoculation of biofilm positive clinical strain of S. aureus through a small hole drilled on the anterolateral wall of the maxillary sinus. For the negative control groups, the animals were only underwent maxillotomy and gelfoam placement, without bacterial inoculation, and the rabbits in the blank control group did not receive any treatment. The maxillary mucosas were harvested at 1,2,4, and 8 weeks postsurgery respectively, and examined with SEM and hematoxylin and eosin (HE) staining to observe the biofilm formation and the exact time of formation. The forth part is efficacy of SF-NS solution against S. aureus bio film-related sinusitis in vivo. Thirty New Zealand white rabbits were randomly divided into five groups, six rabbits in each group. Group A is blank control group, healthy rabbits without any treatment. Group B is model group, the animals in this group were just undergone modeling surgery, but without sinus irrigation. Group C is negative control group (animal models treated with saline). Group D and E were experimental groups (animal models treated with 2×MBC and 5xMBC concentrations of SF-NS solutions respectively). The rabbits of Group B-D were firstly undergone modeling surgery to make the model of S. aureus biofilm-related sinusitis according to the methods described previously. At 4 weeks after the modeling surgery, the rabbits of group A and B were killed and the rabbits of group C-E were undergone a second surgery to indwell irrigation catheters into the right maxillary sinus. The test solutions, SF-NS solution and normal saline, were irrigated slowly into the right maxillary sinus via the indwelling catheter (twice a day,2mL in each time), starting at one day after catheter indwelling surgery. After 7 days of topical irrigation, the sinus mucosa were harvested and examined by SEM, HE staining, RT-PCR and Western-blot for biofilm biomass, histological changes and the expression levels of inflammatory factors. Statistics were analyzed by SPSS16.0.Results:The First Part1 Fluorescence analysis showed that when the AgNO3 concentration was smaller than 8mg/mL in the reaction system of lwt% silk fibroin solution, all the Ag+ ions could be reduced to Ag°by the Tyr residues under light at room temperature.2 When AgNO3 concentration was set to 4mg/mL in the above reaction system, a characteristic peak of silver nanoparticles at about 440nm appeared in the UV-Vis spectra, and the time-resolved UV-Vis spectra showed a rapid growth period from 0 to 20 h and then levels off.3 TEM showed that nanoparticles were formed in the above reaction system and the average size of the particles was about 12±2.1nm. HRTEM confirmed that the resulting products were silver nanoparticles.4 The UV-Vis spectra as well as the appearance of SF-NS solutions were scarcely changed after 30 days of storage either in the dark or in the light.The Second Part1 For biofilm positive bacteria Sa006 and quality control strains ATCC29213, the MIC and MBC of SF-NS were equal and both were 19.2mg/L and 76.8mg/L respectively.2 Congo red experiments showed that the biofilm formation in the medium with increased concentrations of SF-NS, from 1/4×MIC to 1/2×MIC, reduced consistently. When SF-NS concentration increased to MIC, the proliferation of Sa006 was completely inhibited, too.3 CLSM and SEM showed that MIC concentrations of SF-NS had’no effect on the structure and viability of the mature biofilms. When the concentration increased to MBC level or above, the structural damage appeared and correlated with the concentration. When the concentration of SF-NS increased to 5×MBC level, the biofilms were completely destroyed.The Third Part1 All the rabbits in model groups had mucopurulent secretions in the right maxillary sinus at the time of sacrifice. But for the negative control groups, no pus was found in the maxillary sinus.2 CT scans showed the right maxillary sinus of the model rabbits were all filled with soft tissue density. But for the negative control groups, this phenomenon occurred only in the first two weeks.3 HE staining showed that the sinus mucosas of the model rabbits undergone dramatic histological changes and infiltrated with massive inflammatory cells. But for the negative control groups, the mucosal inflammation is mild and quickly returned to normal.4 SEM showed that the model rabbits were all had vary degrees of epithelial damage, and biofilms presented in 100% of rabbits which infected with S. aureus for 2 weeks or more. But for the control groups, the integrity of the epithelium was always presented and no bacterial biofilm had been found.The forth part1 SEM results showed that the epitheliums of group A were intact, and lots of biofilms existed on the destroyed and no ciliated epithelium of group B and group C. For group D, a small amount of BBF could still be seen on the not fully recovered mucosa. But for group E, the epithelial structures were almost recovered to normal and no biofilm had been found.2 HE staining showed that the sinus mucosas of group A were characterized as normal columnar ciliated epithelium without obvious inflammatory cells. The epithelial cells of group B and group C were significantly damaged or missing. In contrast with group B and group C, the epitheliums of group D and group E recovered to some extent, but the infiltration of inflammatory cells did not change significantly.3 At RNA-level, the expression of IL-1β, IL-8, IL-10 and TNF-a of Group B significantly higher than that of group A, and the expression of IL-4, IL-5 significantly reduced. Compared with group B, all the detected cytokines of group C were equal to that of group B, yet group D has a higher level of IL-1β, group E has higher levels of IL-4, IL-5, IL-10 and a lower level of IL-8. Compared with group C, group D has a higher level of IL-1β, group E has higher levels of IL-4, IL-5, IL-10 and a lower level of IL-8. 4 At protein-level, the expression of IL-1(3, IL-8, IL-10 and TNF-a of Group B significantly higher than that of group A. Compared with group B, group C has a lower level of TNF-a, group D has a higher level of IL-1β and a lower level of TNF-α, group E has lower levels of IL-8 and TNF-α. Compared with group C, group D has a higher level of IL-1β, group E has higher levels of IL-1β、IL-10 and lower levels of IL-8 and TNF-α.Conclusions:1 Silk fibroin can be used as a bio-template to produce silver nanoparticles in situ under light at room temperature. This synthetic route is environmental friendly and the resulted silver nanoparticles can be stably stored in usual environment.2 SF-NS is an effective antibacterial agent against S. aureus and can destroy the mature biofilms formed by the same bacterium. This product has a great potential to be used as an antibacterial and anti-biofilm agent in clinic applications.3 The S. aureus biofilm-related animal model of sinusitis established by means of gelfoam placement and biofilm-positive S. aureus innoculation through the hole drilled on the anterior wall of the rabbit’s maxillary sinus is simple and reliable.4 S. aureus biofilms induced a bilateral-biased immune response within the sinus mucous which characterized by high levels of Th1 cytokines and low levels of Th2 cytokines. SF-NS solution is an effective topical nasal anti-biofilm agent which can not only remove the biofilms, but also promote the epithelium repair and the resolution of inflammation. |
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