Biocompatibility of a new silver-polysaccharide nanocomposite system (chitlac n-Ag) in human gingival fibroblasts/S. mitis coculture

BisGMA/TEGDMA thermosets are biomaterials used for restorative dentristry, but bacteria are responsible for a negative outcome. To avoid bacterial biofilm formation on dental devices and to reduce cytotoxicity against eukaryotic cells, a new material with antimicrobial properties was developed. Silver nanoparticles, with antimicrobial properties, were stabilized with a polyelectrolyte solution (Chitlac) and used to coating methacrylic thermosets. The aim of this study was to evaluate the ability of human gingival fibroblasts (HGFs) cultured alone or with Streptococcus mitis to adhere on this nanocomposite system with antimicrobial properties. HGFs obtained from fragments of healthy marginal gingival tissue, were seeded on Chitlac or Chitlac n-Ag thermosets and then co-cultured with S. mitis for 48 h. In order to mimic the microenvironment of oral cavity, when indicated, saliva was added, to adhere on this nanocomposite system with antimicrobial properties. HGFs obtained from fragments of healthy marginal gingival tissue, were seeded on Chitlac or Chitlac n-Ag thermosets and then co-cultured with S. mitis for 48 h. In order to mimic the microenvironment of oral cavity, when indicated, saliva was added. Cell morphology, adhesion and biofilm formation were evaluated by means of Scanning Electron Microscopy (SEM); collagen type 1 secretion was verified by ELISA assay; beta 1 integrin and PKC alpha expression were detected by immunofluorescence analysis. SEM images show that fibroblasts (with or without bacteria) spread and adhere on Chitlac or Chitlac n-Ag thermosets, maintaining the typical elongated morphology, while bacteria amounts and biofilm decrease on Chitlac n-Ag thermosets. Collagen type 1 secretion assay indicates a lower production of collagen by the cells seeded on both kind of thermosets compared to control, while in the presence of bacteria on Chitlac n-Ag thermoset the amount of type 1 collagen is very low compared to control and to Chitlac thermoset. When saliva is added to the cells increases the collagen secretion mainly in the co-culture on Chitlac n-Ag thermoset in whic the value is similar to the control. Confocal images clearly show that cells are tigthly attached on both kind of thermosets and beta 1 integrin appears well organized especially in cells seeded on Chitlac n-Ag thermoset. Confocal analysis of PKC alpha indicates that the protein, in the presence of saliva, translocates to the nucleus, while in the other samples PKC alpha expression appears cytoplasmic and less intense. Thus, in our experimental system, the activation of PKC alpha/beta1 integrin intracellular signalling confirms a key role played by oral bacteria and saliva in the adhesion process that occurs in vivo in HGFs seeded on both kind of thermosets and suggests this new biomaterial useful for the restorative dentistry.