The aim of this study will be to propose the synthesis and characterization of a nanocomposite based on silver nanoparticles and ²-calcium glycerophosphate nanoparticles (Ag/²-Calcium glycerophosphate), evaluate the antimicrobial effect of this nanocomposite on Candida albicans (ATCC 10231) e Streptococcus mutans (ATCC 35668) and assess its influence on human dental pulp cell in the presence and absence of C. albicans e S. mutans biofilms. First, colloidal silver nanoparticles (5 nm) will be synthesized by reduction of the silver nitrate through sodium citrate. ²-Calcium glycerophosphate nanoparticles will be synthesized by grinding conventional particles of ²-Calcium glycerophosphate. These two compounds will be associated seeking a concentration of 9 wt% of phosphate. During the nanocomposite synthesis X-rays diffraction and transmission electronic microscopy will be performed. Subsequently, according to the microdilution method, it will be determined the minimum inhibitory concentration of Ag/²-Calcium glycerophosphate capable of inhibiting the growth of C. albicans and S. mutans in the planktonic and sessile state. After, the effect of Ag/²-Calcium glycerophosphate on preformed biofilms will be assessed through the metabolic activity of the cells. Moreover, human dental pulp cells will be obtained from recently extracted teeth at Surgery Clinic of Araçatuba Dental School - UNESP, primary cultures (n=3) will be stablished through an explant technique and after the fourth passage the cells will be used in the assays. C. albicans and S. mutans biofilms will be formed (24 h) on inserts and used in co-culture model dental pulp cells/biofilm of both microorganisms. The effect of different concentrations and periods of treatments with nanocomposite on the human dental pulp cells, in the presence and absence of biofilms will be evaluated through 1) analysis of cell proliferation by MTT, 2) expression of alkaline phosphatase, dentin sialophosphoprotein, dentin matrix protein 1, osteocalcin and collagen type I by real-time PCR, 3) alkaline phosphatase activity assessment by thymolphthalein release by hydrolysis of thymolphthalein monophosphate, 4)detection and quantification of mineralization by alizarin red staining. Controls devoid ²-calcium glycerophosphate and with calcium hydroxide will be included in all analyzes, and all tests will be performed in triplicate on three different occasions. Data will be analyzed to employ the most appropriate statistical test. P< .05 will be considered statistically significant.
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