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Biofilms Developed on Dental Implant Titanium Surfaces with Different Roughness: Comparison Between In Vitro and In Vivo Studies


Microbial biofilms developed on dental implants play a major role in perimplantitis’ pathogenesis. Many studies have indicated that surface roughness is the main feature favoring biofilm development in vitro, but its actual influence in vivo has still to be confirmed. In this study, the amount of biofilm formed on differently treated titanium surfaces, showing distinct roughness, has been examined both in vivo and in vitro by Confocal Laser Scanning Microscopy. In vitro studies availed of biofilm developed by Pseudomonas aeruginosa or by salivary bacteria from volunteer donors. In vivo biofilm production was obtained by exposing titanium discs to the oral cavity of healthy volunteers. In vitro experiments showed that P. aeruginosa and, to a lesser extent, salivary bacteria produce more biomass and develop thicker biofilms on laser-treated and sandblasted titanium surfaces with respect to machined ones. In vivo experiments confirmed that bacterial colonization starts on sites of surface unevenness, but failed to disclose biomass differences among biofilms formed on surfaces with different roughness. Our study revealed that biofilm developed in vitro is more easily influenced by surface features than biofilm formed by complex communities in the mouth, where the cooperation of a variety of bacterial species and the presence of a wide range of nutrients and conditions allow bacteria to optimize substrate colonization. Therefore, quantitative differences observed in vitro among surfaces with different characteristics may not be predictive of different colonization rates in vivo.

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The authors would like to thank GEASS s.r.l (Pozzuolo del Friuli, Udine) for donating some of the materials used in this study. CLSM images were generated in the Optical Microscopy Center of the University of Trieste at the Life Sciences Department, funded as detailed at “” Special thanks are due to the facility manager, Dr. Gabriele Baj, for his expert advice.

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Correspondence to Lucilla Dolzani.

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The authors declare that they have no conflict of interest.

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Supplementary Figure 1 Acrylic splint carrying titanium discs (TIF 1938 KB)


Supplementary Figure 2 CLSM image of a LT titanium surface covered with biofilm developed after 1-day exposure to the oral environment (magnification: 60x) (TIF 12400 KB)


Supplementary Figure 3 Biofilm developed on glass slides by P. aeruginosa (a) and Mixed Salivary bacteria (b). Staining: Hucker crystal violet; magnification: 600x. Method: sterile standard microscopy glass slides with self-adhesive 12 wells silicone gaskets (Ibidi) were used. 200 µl of BHI containing either 5x105 bacteria from a pure P. aeruginosa culture or 5 µl of MSB were dispensed into each well. Slides were covered with the supplied lid and incubated for 48 hours at 37°C to allow biofilm development. Each well was then washed 3 times with sterile saline, the gasket was removed and biofilm was fixed by exposure at 60° for 1 hour. The slide was stained for 15 min with Hucker crystal violet and observed under the light microscope (TIF 8274 KB)

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Bevilacqua, L., Milan, A., Del Lupo, V. et al. Biofilms Developed on Dental Implant Titanium Surfaces with Different Roughness: Comparison Between In Vitro and In Vivo Studies. Curr Microbiol 75, 766–772 (2018).

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