Introduction: Since ancient times, silver has been believed to control infections and silver based medical products have been proven to retard and prevent bacterial growth. In the field of dentistry, the use of silver ions and nanoparticles has been explored to counteract bacteria in resins and implants, as silver can destroy bacterial cell walls by reacting with sulfhydryl groups on membranes proteins. However, it is also cytotoxic to eucaryotic cells, wich are capable of internalizing nanoparticles. In this work, we investigated Chitlac-nAg, a colloidal solution of silver nanoparticles stabilized with a lactose-modified chitosan. We examined the biological effects of Chitlac-nAg treatment (24-48 h) on a co-culture system, made of primary human gingival fibroblast (HGFs) and Streptococcus (S.) mitis in the presence of saliva, developed to mimic the microenvironment of the oral cavity. We sought to determine its biocompatibility and its efficiency in counteracting oral hygiene-related diseases. Methods: Cytotoxicity was evaluated by MTT test, LDH release and annexin-V/PI apoptosis assay; reactive oxygen species (ROS) production, lysosome metabolism and nanoparticle uptake was investigated by flow cytometry. Light and electron microscopy images confirm the nanoparticle uptake. Collagen expression by western blotting and IL-6 and PGE2 release by ELISA were also assessed. Moreover, LC3-II expression, a well known autophagy marker, was recorded by immunofluorescence. Results: In vitro results show that after 24 h of treatment, Chitlac-nAg increases LDH release, apoptosis occurence, ROS production and IL-6 and PGE2 production, with a major extent in samples with S. mitis and/or saliva. However, after 48 h, Chitlac-nAg seems not to exert cytotoxicity anymore, while data from light and electron microscopy point out chaanges in cell side scatter, LC3-II expression and lysosome compartment modification suggesting that HGFs could be able to promote cell survival through a homeostasis mechanism involving autophagy. Conclusion: After an early cytotoxicity exterted by Chitlac-nAg on HGFs, the presence of silver nanoparticles, saliva and bacteria, seems to induce HGFs to activate a homeostasis mechanism promoting cell survival through autophagy. Basing on its good biocompatibility we suggest this new tool useful for the realization of dental devices.
Cell protection mechanism through autophagy in a co-culture of human gingival fibroblasts and Streptococcus mitis treated with Chitlac-nAg
Monica Rapino;
2015
Abstract
Introduction: Since ancient times, silver has been believed to control infections and silver based medical products have been proven to retard and prevent bacterial growth. In the field of dentistry, the use of silver ions and nanoparticles has been explored to counteract bacteria in resins and implants, as silver can destroy bacterial cell walls by reacting with sulfhydryl groups on membranes proteins. However, it is also cytotoxic to eucaryotic cells, wich are capable of internalizing nanoparticles. In this work, we investigated Chitlac-nAg, a colloidal solution of silver nanoparticles stabilized with a lactose-modified chitosan. We examined the biological effects of Chitlac-nAg treatment (24-48 h) on a co-culture system, made of primary human gingival fibroblast (HGFs) and Streptococcus (S.) mitis in the presence of saliva, developed to mimic the microenvironment of the oral cavity. We sought to determine its biocompatibility and its efficiency in counteracting oral hygiene-related diseases. Methods: Cytotoxicity was evaluated by MTT test, LDH release and annexin-V/PI apoptosis assay; reactive oxygen species (ROS) production, lysosome metabolism and nanoparticle uptake was investigated by flow cytometry. Light and electron microscopy images confirm the nanoparticle uptake. Collagen expression by western blotting and IL-6 and PGE2 release by ELISA were also assessed. Moreover, LC3-II expression, a well known autophagy marker, was recorded by immunofluorescence. Results: In vitro results show that after 24 h of treatment, Chitlac-nAg increases LDH release, apoptosis occurence, ROS production and IL-6 and PGE2 production, with a major extent in samples with S. mitis and/or saliva. However, after 48 h, Chitlac-nAg seems not to exert cytotoxicity anymore, while data from light and electron microscopy point out chaanges in cell side scatter, LC3-II expression and lysosome compartment modification suggesting that HGFs could be able to promote cell survival through a homeostasis mechanism involving autophagy. Conclusion: After an early cytotoxicity exterted by Chitlac-nAg on HGFs, the presence of silver nanoparticles, saliva and bacteria, seems to induce HGFs to activate a homeostasis mechanism promoting cell survival through autophagy. Basing on its good biocompatibility we suggest this new tool useful for the realization of dental devices.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
