## 4 Discussion  
In this present study, we have demonstrated not only topographic features of Ti implant surfaces, but also how their chemical composition may affect their microbiological properties. The topographic characterization of tested implant surfaces was carried out using multiple analysis parameters and appropriate filtering to obtain an exhaustive description of their texture and reduce the errors due to form and waviness, as suggested by [PERSON] and [PERSON] (2000) [16]. Following the classification of implant surfaces proposed by these authors, both T and MA tested surfaces can be considered \"smooth\" (Sa < 0.5 \(\upmu\)m). Of note, most of the implants on the market, including those roughened by acid-etching techniques, have a minimally rough (Sa 0.5-1.0 \(\upmu\)m) or moderately rough (Sa 1.0-2.0 \(\upmu\)m) surface [17]. Nevertheless, MA surfaces showed significantly higher Sa values than T ones, whereas the chemical composition of tested specimens (grade 4 vs. grade 5 Ti), did not seem to affect surface topography. The low roughness values found for MA samples could be due to the mild organic acids (e.g., oxalic and maleic acid) used for their treatment, resulting in a surface texture characterized by a sequence of multiple pits and ridges less pronounced than those of strong acids conventionally employed (e.g., phosphoric acid, hydrochloric acid) (see Figure 1). The use of in vitro models in studies on microbial adhesion allows reproducibility, cost-effectiveness, and control of possible influencing variables. On the other hand, the translation of in vitro results to clinical reality is more difficult. In this sense, ex vivo experimental models offer the chance to investigate these phenomena in conditions closer to the clinical ones, with a heterogenous biofilm, composed of a variety of symbiotic microorganisms, with the presence of an acquired pellicle of salivary origin, and with the detaching forces impressed by the salivary flow and the functional action of peri-oral structures [18]. The microorganism chosen for the in vitro part of the present study was the _S. sanguinis_, a common resident of the oral cavity, due to its role of initial colonizer of the human oral biofilm [19]. The sonication protocol for bacteria detachment was preferred among other available techniques (e.g., scraping) due to its high efficiency and recovery rate, as previously demonstrated [20]. Incubation times of 2 and 6 h were chosen in the present study to investigate in vitro the initial phase of bacterial adhesion on implant  
Figure 5: Bacterial adhesion on turned (T), mildly acid-etched (MA), grade 4 and grade 5 Ti implant surfaces after 12 h ex vivo staying in the mouth. Data are expressed as counts of Colony Forming Units (CFU) on a logarithmic scale (median, interquartile range, minimum/maximum). N = 12. ** = \(p\) < 0.01; *** \(p\) < 0.001.  
surfaces. On the other hand, a longer time (12-24 h) is needed using ex vivo study models to obtain a structured biofilm upon Ti surfaces [18; 21]. We found in vitro a significantly higher CFU count of _S. sanguinis_ on MA compared to T samples after 2 and 6 h of incubation. The absolute values of all groups tend to increase over time, whereas the inter-group difference tends to markedly decrease from 2 to 6 h, although maintaining the statistical significance. In a longer period of observation, in the ex vivo experimentation, such a difference was even more pronounced than in vitro, although CFU absolute values ex vivo were lower than those found in vitro. CFU values were found about 10-fold higher on MA than T surfaces and, furthermore, 2 to 3.5-fold higher on grade 5 than grade 4 surfaces. It must be underlined that a direct comparison between in vitro and ex vivo results at the different time points cannot be made due to the significantly different experimental conditions applied, and that several environmental parameters potentially affecting bacterial adhesion (e.g., pH, composition, etc.)