To confirm the promising photocatalytic antibacterial and cytocompatibility effects of a coating developed in our previous study (Nagay et al. 2019) for dental implants application, the aim of the present study is to evaluate in vitro, in situ and in vivo the antibacterial, immunogenic and osteogenic activity of the TiO2 coating codoped with nitrogen (N) and bismuth (Bi) via plasma electrolytic oxidation (PEO) of commercially pure titanium surface (cpTi), in the presence and absence of visible light. CpTi discs (10 mm in diameter × 1 mm thick) and implants (1.6 mm in diameter × 3 mm in height) will be subjected to different surface modifications: sandblasting with aluminum oxide followed by acid etching (control); and modified by PEO with surface of TiO2 coating codoped with N and Bi (Bi-TiO2, experimental). Three experiments will be conducted: (1) In vitro study, to evaluate the release of metal ions, the antimicrobial potential (microcosm model considering saliva of volunteers as inoculum) after application or not of visible light, and biocompatibility (pre-osteoblastic cells) of the coating developed on the discs. (2) In situ study, where the volunteers will use for 7 days a palatal appliance containing 3 control discs and 3 experimental discs, to allow the accumulation of biofilm, which will be irradiated by visible light or not (dark). Colony-forming unit (CFU) count and biofilm analysis by CLSM will be performed immediately and after 24 h of light application. (3) In vivo studies, to verify the osseointegration potential of PEO-treated implants containing Bi-TiO2 on the surface compared to control after implantation in rats tibiae. Histological and immunohistochemical analyzes for decalcified tissues, and micro computed tomography (micro-CT), bone-implant contact, newly formed bone area, reverse torque and osteogenic gene expression for calcified tissues will be performed. To verify the in vivo antibacterial effectiveness and inflammatory response of the surface treatment with and without the light application, discs (5 mm in diameter × 1 mm in height) with control and experimental surfaces will be implanted on dorsal region of rats. After in situ biofilm formation on the surfaces, 1 disc (control or experimental surfaces, with or without previous light application) will be installed in each animal. Colony-forming unit (CFU) count and cytokine analysis (IL-1 beta and TNF-alpha) will be performed. Quantitative data will be submitted to the most appropriate statistical analysis with 5% significance level.
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