## 4 Discussion  
The production of metallic implants and biomedical tools with the help of 3D printing has spread considerably in recent years. However, the main cause of failures, both early and late, of orthopedic prostheses, dental implants, and membranes for guided bone regeneration is bacterial colonization [26]. Current research in dentistry and biomedical  
Figure 6: SEM observations of the different surfaces covered by the bacterial biofilm: machined (3900\(\times\)**A**, 6100\(\times\)**E**), DAE (3900\(\times\)**B**, 6100\(\times\)**F**), and 3D (3900\(\times\)**C**, 6100\(\times\)**G**). The average values of the colony forming units (log10)/mL (**D**) and biofilm mass formation (**H**) of \(S\). _oralis_ at 48 h (error bars = +/ \(-\) standard deviation). * \(p\) significant at 0.05. Figure 7: Representative images of live/dead staining of _Streptococcus oralis_ biofilm growth on different titanium surfaces: machined (**A**), DAE (**B**), and 3D (**C**) samples and positive controls (**D**). Average percentage of viable (green) and dead (red) cells on different groups (**E**). Error bars = +/ \(-\) standard deviation. * \(p\)-value < 0.01.  
devices aims to find novel materials and technologies able to counteract the bacterial growth but at the same time to accelerate the healing of infected sites or to promote the bone osseogeintegration of dental implants and bone prosthesis [14; 27; 28]. The novel surfaces are manufactured and designed with a high porosity in order to increase interaction with cells, and to promote accelerated healing and osteointegration. The 3D sample was chosen from a group of other titanium ones, produced by SLM and tested on a previous study [21]. In particular, it showed not only biocompatibility but also the ability to increase the Dental Pulp Stem Cells (DPSCs) proliferation paralleled by the decrease in LDH released in a time-dependent manner [21]. The 3D surface showed a behavior resembling the inner structure of native bone, allowing cells to better adhere inside the specimen, with proteins related to cell adherence being highly expressed. It also showed osteoconductive properties, with the profile of osteogenic markers being improved compared with titanium samples [21]. However, once that pioneer bacteria colonized a biomaterial, the procedure for bacterial removal could be very difficult, especially for the ability of the biofilm to protect bacteria against antibiotics, so novel treatments have been proposed and studied [28]. Consequently, the introduction of novel materials and surfaces that could contrast the adhesion of bacterial pioneers, such as _Streptococcus oralis_, could represent a real advancement for the prevention of this disease [14; 15]. In this study, _S. oralis_ was used because it is an early colonizer during the process of biofilm formation on implant surface and provides the basis for the subsequent colonization of facultative and obligate anaerbes. The novel 3D surface, investigated in this study, had the advantages of being manufactured through SLM, so a tridimensional geometry was computer-projected in order to promote cellular interactions and osseointegration. This 3D surface showed very impressive results against _S. oralis_, especially when compared with machined discs. The 3D one was characterized by high nano-roughness and oxygen content in the upper layers and a good micro-roughness that presumably contributed in the reduction in bacterial CFUs, as previously found in the literature [14; 15]. Indeed, as suggested by [PERSON] et al., the presence of nano-roughness could act as a spacer between titanium and bacteria, which are characterized by a rigid structure [29]. Another difference between 3D discs and the other groups is the chemical composition; as shown in the EDX analysis, the 3D contained a certain percentage of aluminum because it was manufactured with a Ti6 Al4V alloy contrary to the machined and DAE samples, which were in pure Ti.