Effect of titanium dioxide nanotubes on biological properties of conventional glass ionomer cement

Abstract

Titanium dioxide nanotubes (n-TiO2) have been applied to dentistry to improve general properties of restorative materials. Our group has recently demonstrated that n-TiO2 may improve compression and wear resistance of conventional Glass Ionomer Cement (GIC), increase GIC hardness without affecting surface roughness, dentin micro shear-bond-strengh, and cell morphology as compared to GIC alone (Grant FAPESP #2016/13786-0). However, the ability of n-TiO2 to modulate bacteria and host response remains to be defined. Thus, the goals of the present study iare to: 1. define the impact of n-TiO2 on the antimicrobial properties of GIC; and 2. determine the potential of n-TiO2 to modulate the expression of immuno-inflammatory markers by fibroblasts (NIH/3T3 cells) treated or not with Fusobacterium nucleatum (Fn) total extract. In order to achieve those goals, n-TiO2 will be added at different concentrations (0%, 3%, 5%, 7% by weight) to GIC matrix (Ketac Molar EasyMix) and the following experiments performed: 1. Agar diffusion test (n=6): to determine a potential antimicrobial capacity of GIC, with or without, n-TiO2, bacteria inoculum (S. mutans or L. acidophilus) will be used, and chlorhexidine (0.12%) and distilled water will be used as positive and negative controls, respectively; 2. Cell viability (n=6): to assess the impact of GIC, with or without n-TiO2, on bacteria cell viability, 1x108 bacteria CFU/mL will be cultured in the presence of the tested materials and cell viability will be determined using the Live/Dead baclight fluorescence technique. Number of viable and non-viable cells will be obtained as the proportion of the total area of the disks in three random regions using the ImageJ program; 3. Cell morphology (n=6): the effect of tested materials on cell morphology will be defined by scanning electron microscopic analysis (SEM) (15 KV, 2000X), 4. Bacteria gene expression analysis (n=6): a potential impact of GIC, with or without n-TiO2, on transcript levels of vicR, covR, gtfB, gtfC, and egtfD in S. mutans and slpA, slpB e slpX for L. acidophilus will be determined by real-time quantitative PCR reactions; 5. Protein expression (n=6): NIH/3T3 cells will be cultured on GIC disks, with or without n-TiO2 (12 and 18 h), treated or not with Fn total extract (20, 2 and 0.2 ¼g/mL) and IL-1², IL-10, IL-6 and TNF secreted levels will be determined by mutiplex analysis using the MagPix system; and 6. Gene expression analysis in NIH/3T3 cells (n=6): IL-1², IL-10, IL-6, TNF transcript levels will be determined in NIH/3T3 cells cultured on GIC disks, with or without n-TiO2, treated or not with Fn total extract (3, 6 and 18 h). Experiments will be repeated, at least twice with three biological replicates, and in general, experimental times will be 1, 3 and 7 days (unless otherwise stated). Data were submitted to statistical analysis, considering the 5% level of significance (± = 0.05). (AU)