# Fabrication of a Promising Hierarchical Porous Surface on Titanium for Promoting biocompatibility
## 3 Results and Discussion
### Cellular Behaviors  
Figure 5 depicts the SEM micrographs of MG-63 cells on the investigated specimens after cultured with 1 day. The cells appear in a spindle-like shape and the centrally protruding nuclei were surrounded by thin cytoplasmic edges on the Ti specimen as shown in Figure 5a. As the incubation period increasing, it was found that the cells have almost completely adhered to the specimen surface. A similar cell  
Figure 4: Dark-field TEM micrograph of nano-particle taken from the surface layer of the HFP-Ti specimen at 1 A/dm\({}^{2}\) for 10 min.  
Figure 3: TF-XRD patterns of the investigated specimens (Ti, HFP-Ti at 2 A/dm\({}^{2}\) for 10 min, and HFPA-Ti).  
adhesion behavior can also be observed on the HFP-Ti specimens with different pretreatment current densities. As the HFPA-Ti specimen underwent an incubation of 1 day (Figure 5b), it is clearly seen that the cells exhibited good adhesion behavior with numerous filopodia (as indicated by black arrows) on the nanoporous surface from a higher magnification observation. The filopodia of cells tightly grabbed the nanoporous surface in comparison to the cells only adhered to the Ti and HFP-Ti specimens. This characteristic of cell adhesion behavior can also be found on the investigated specimens with a longer incubation period. Therefore, it is evident that the HFPA-Ti specimen possessed great potential to enhance cell adhesion ability.  
It is well-known that hydrogen penetrated the samples during their mechanical polishing or chemical etching [29]. Hydrogen was also detected on the subsurface of the mirror-polished and sandblasted (with large grit and acid (SLA)) samples [30]. In the present study, the HFP-Ti specimens exhibited the hydrogen reaction and/or penetration. Hydrogen existed on the surface, and the concentration of hydrogen increased by increasing current density. The amount of hydrogen increased for the formation of a large number of nano-\(\gamma\)-TiH. Hence, the surface formed an oxide layer with microporous structure, as shown in Figure 1b. While the oxide layer acted as the barrier layer for hydrogen diffusion and blocked the hydrogen desorption. The ability of the oxide layer to block the metal ions diffusion can be promoted by raising the oxide thickness through surface treatment. As the Ti oxide layer thickness increased, the adsorption ratio of albumin/fibrinogen of Ti enhanced by six times [31]. However, the HFPA-Ti surface layer showed a hierarchical porous (micro and nanoporous)  
Figure 5: SEM micrographs of MG-63 cells on the investigated specimens after cultured with 1 day: (**a**) Ti and (**b**) HFPA-Ti.  
surface as shown in Figure 1c,d. The formation of the hierarchical porous surface could be attributed that the hydrogen absorption and charging in the HF pretreatment caused the \(\alpha\)-Ti to nano-\(\gamma\)-TiH transformation. After the formation of nano-\(\gamma\)-TiH, nanopores are generated by anodization to dissolve nano-\(\gamma\)-TiH. The formation of the nano-\(\gamma\)-TiH phase or the hydrogen absorption is essential to produce a nanoporous TiO\({}_{2}\) layer after the anodization treatment. Moreover, the anodization treatment results in the variation of enthalpy (\(\Delta\)H \(>\) 0). The elevated electrolyte temperature is beneficial to the reactants. An increase in the electrolyte temperature inhibits the oxide films formation. This is because of a controllable anodized oxide films thickness [32]. The oxide layer formation can mainly be attributed to redox reactions. The hydrogen reaction and penetration cause the nano-\(\gamma\)-TiH formation. It is believed that the nanophases dissolution is crucial for the nanoporous Ti oxide layer formation. Thus, Ti implants with a hierarchical porous (micro and nanoporous) TiO\({}_{2}\) surface can promote cell adhesion, thereby enhancing the bone cell ingrowth into the nanoporous structure, which provided the mechanical fixation of the implant to the bone.