The Broad-spectrum Antimicrobial Activity And Biocompatibility Of The Mesoporous Silica Nanoparticles Encapsulated With Silver Nanocrystals In Vitro

Part I The Broad-spectrum Antimicrobial Activity of the Mesoporous Silica Nanoparticles Encapsulated with Silver NanocrystalsIntroduction:The Mesoporous Silica Nanoparticles Encapsulated with Silver Nanocrystals (Ag-MSN) material has an excellent biocompatibility and mechanical capacity. It presents an ability to load and control the release of drugs, therefore, it is a potential antibacterial coating for bone substitute materials.Purpose:In this study we examined the Ag-MSN material’s antibacterial capacity in vitro and its Ag ion release characteristics to evaluate the feasibility for the use of this material to treat the bone defect after chronic osteomyelitis surgery.Methods:The ex vivo antibacterial effect against Staphylococcus aureus (S. aureus,25923, ATCC), Escherichia coli (E. Coli,25922, ATCC), Pseudomonas aeruginosa (P. aeruginosa,9027, ATCC), Bacteroides fragilis (B. fragilis,25285, ATCC), and Candida albicans (C. albicans,10231, ATCC) of different concentration of Ag-MSN suspension was detected by Standard broth dilution method and OD600Bacterial growth curve assay. To verify the Ag ion release curve and assess the material’s Ag release characteristics, the Ag-MSN (400/200/100/5Oμg/ml) was added into simulated body fluid (SBF) and the Ag concentration was determined by flame atomic absorption spectrometry (FAAS) at different time points.Result:We confirmed that the Minimum Inhibitory Concentration (MIC) of Ag-MSN material against S. aureus, E. Coli,P. aeruginosa and B. fragilis were below200ug/ml, and the Minimum Bactericidal Concentration (MBC)were at400,200,200,200ug/ml, respectively. The middle concentration of Ag-MSN suspensions could significantly inhibit bacterial growth compared with the control group after12hours, however, the Ag-MSN suspension did not exhibit obvious sterilization effect on C. albicans, and could only inhibit its growth at400μg/ml concentration. We observed a similar trend of Ag ion release in different concentration groups of Ag-MSN suspensions that the Ag ion concentration of all groups reached the peak after soaking for6hours, and stabilized after soaking for3days, which had no significant difference compared to soaking for14days. Ag ion’s biological safety Concentration was reported to be54mg/L, whereas its minimal inhibitory concentration was0.5mg/L—0.05mg/L, the Ag ion in400,200and100μg/ml concentration groups were always between these two levels during the experiment.Conclusion:Ag-MSN material has a significan bactericidal effect against S. aureus, E. Coli, P. aeruginosa, and B. fragilis in vitro, also it presents an inhibitory effect against C. albicans. These data demonstrate that the Ag-MSN material has a good sustained release quality in SBF, which makes it a potential candidate of bone substitute antibacterial material against chronic osteomyelitis. Part II The Mesoporous Silica Nanoparticles Encapsulated with Silver Nanocrystals’Influence on Proliferation and Differentiation ability of OsteoblastsPurposes:In this study we examined the Ag-MSN and MSN’s biocompatible in vitro and their Influences on Proliferation and Differentiation ability of OsteoblastsMethods:The CCK8assay was used to test the biocompatibility of different concentration of Ag-MSN and MSN dissolution while Osteoblast-like MG63cells were cultured. The alkaline phosphatase (ALP) and the bone gla protein(BGP)’s activity were tested by ELISA method,while their gene expressions were tested by qRT-PCR method.Result:The CCK8assay indicated that the Ag-MSN and MSN didn’t restrain the proliferation of MG63cells. The results of alkaline phosphatase (ALP) and bone gla protein(BGP) activity test and the expression of osteogenic marker genes, such as ALP and BGP, demonstrated that the differentiation of MG63cells might be enhanced by Ag-MSN and MSN.Conclusion:Ag-MSN and MSN all have good biocompatibility, the Ag-MSN dissolution and the MSN dissolutions can all enhance the differentiation of MG63cells, Ag-MSN is a safe and potential candidate of bone substitute antibacterial coating material against chronic osteomyelitis. Part III The Mesoporous Silica Nanoparticles Encapsulated with Silver Nanocrystals’Influence on OPG/RANKL Signaling pathwayPurposes:To discuss the influence and possible mechanisms of the different concentration of Ag-MSN and MSN dissolution on OPG/RANKL Signaling pathway by testing the related gene expressions and their protein synthesis of osteoblastsMethods:The intracellular OPG and RANKL’s gene expressions were tested by qRT-PCR method, while the MG63cells co-cutured with different concentration of Ag-MSN and MSN dissolution for1/3/7days. Their protein activity were tested by Western Blot method.Result:The result of qRT-PCR method shows that OPG gene expression quantity increased since3days, then has been maintained at a higher level,while RANKL gene expression quantity increase since co-cuture7days, no statistical difference had found between the experimental group and control group (P>0.05).The western Blot result shows that the osteogenesis OPG protein in the sample cell has no obvious difference between experimental groups and control group, but the experimental groups’RANKL protein showed a moderately elevation compared with control group, expecially the groups with high concentration (400ug/ml Ag-MSN groups and400ug/ml MSN group).Conclusion:Ag-MSN and MSN had no significant impact on the OPG/RANKL pathway in differentiating osteoblast like cells, but MSN and Ag-MSN of high concerntrations could promote the well-developed osteoblast like cells’expression of RANKL, thereby activating osteoclast moderately. However, further studies are needed to determine whether Ag-MSN and MSN could enhance the funtion of osteoclast in vivo so that cause adverse effects of new-born bone destruction.