## 4 Discussion  
New technologies and research in the dental implant field have led, in the last decade, to a higher success rate and a better understanding of osteointegration processes [29]. Progress mainly derived from new implant surface and, consequently, the introduction of devices with different roughness, wettability, and chemistry [30]. These variables are strongly related, and understanding their role in the osteointegration process has given implants superior clinical outcomes. The topography of the surfaces has been developed at the micro (1-10 \(\upmu\)m) and nano level (1-100 nm). Micro-roughened surfaces are obtained with SLA or anodic oxidation treatments, while nano-roughened surfaces derived from TiO\({}_{2}\) nanotube layer, Coating with Hydroxyapatite or Calcium-Phosphorus Compound or Functional Peptides, Photofunctionalization, Laser Ablation, and Flouride Treatment by Cathodic Reduction [31]. HNS surface showed encouraging results in vitro [32]. Several studies suggest that nanostructured topography provides a structure analogous to natural bone enhancing protein absorption and the signaling pathways controlling cells adhesion, proliferation, and differentiation [33,34]. Surface wettability is another crucial aspect of osseointegration [35]. Wettability is quantified by the Contact Angle (CA), and hydrophilic, hydrophobic, superhydrophobic, or superhydrophobic surfaces are recognized. Most in vitro studies highlight those hydrophilic surfaces enhance early osteointegration due to close interaction with biological fluids and better protein adsorption [36,37]. However, some studies suggest that extremely high surface energies can hinder motility and cell functions [38]. Implant chemistry is modified by means of different coating materials, including carbon, bisphosphonates, bioactive glass and ceramics, fluoride, hydroxyapatite and calcium phosphate, Ti-nitride and salts [39]. In this study, a new surface created by adding salts to an SM surface (HNS) was tested. Salt's addition was made in order to improve the wettability and stabilize the hydrophilic surfaces over time, avoiding wet storage [40]. It is quite interesting to note that SM surface determines a slight but significant increase in cell adhesion in a short time compared to HNS surface. It has been observed that large increases in cell adhesion could also correlate with increases in cell spreading at an early time. In this study, we observed a slight difference in the level of spreading, which is greater at T2 than at T1, with cells seeded on HNS surfaces appearing very slightly more elongated and less round than those seeded on SM surfaces indicating faster differentiation on HNS surfaces. HNS surface has long and intertwining filopodia that could be a response of cells to surface nano-topography that results in a higher stage of differentiation. This could indicate that in the SM surface there is a greater proliferation and cell adhesion which is not an aspect that implies a faster differentiation. As mentioned above, cell morphological differences are much more evident 24 h after seeding. It is possible to observe that cells on the SM surface are rounder, with greater cell spreading and that these cells have a tendency to aggregate more than those on the HNS surface. Although it is necessary to deepen with further studies, this phenomenon could be related to the tendency to proliferate more on this surface. Indeed, the proliferation essay showed statistically significant differences between HNS and SM at 48 h and 120 h, with SM surface greatly enhancing proliferation. Although the precise mechanism is still unknown and further studies are needed, these results could also be due to the residual impurity and contaminants on HNS surface hindering proliferation despite its ideal topography and hydrophilicity. The difference between HNS and SM can be also attributable to in vitro experimental conditions, and other studies are needed to clarify the role of Bioactivation with salt in the early stages of healing.