# Anodic Oxidation of 3D Printed Ti6 Al4V Scaffold Surfaces: In Vitro Studies
## 3 Results and Discussion
### In Vitro Studies
#### 3.2.4 Cell Morphology  
The present in vitro study demonstrated that the surface treatment (anodic oxidation) on the Ti6 Al4V scaffolds affected cell morphology. Cell attachment, spreading, and subsequent proliferation are closely related to surface properties such as composition, roughness, wettability, and morphology. After anodic oxidation, it was expected that the chemical components and nanotopography on the formed layer would increase the adsorption of proteins on the surface, thereby promoting cell adhesion and proliferation. In Figure 11, the surface of the scaffolds (as built, ac, and ao) with the morphology of Saos-2 cells cultured on Day 1 is shown. Image analysis showed that cells attached to the surfaces were quite different. The cells on the as-built scaffold surfaces adhered firmly to particles with a round shape (Figure 11a), and the same morphology was observed in scaffolds with chemical polishing, but with fewer adhered cells (Figure 11b). In contrast, the cells on anodized surfaces exhibited a full-bodied and polygonal osteoblastic shape, with filopodia used to attach to the scaffold (Figure 11c). At high magnification (Figure 11d), it is possible to observe pseudopods in multiple contact points with tiny protrusions extending from the lamellipodia on the anodized surface. This behavior was confirmed in the ALP analysis on Day 1. Figure 10: Alkaline phosphatase (ALP) activity. ALP activity of Saos-2 cells assessed during 14 days of culture on top of Ti6 Al4V scaffolds (\(\delta\) represents statistically different from Day 14; \(\varepsilon\) represents statistically different from Day 7; \({}^{*}\) represents statistically different from the indicated group). However, as seen in the ALP assays (Figure 10), the adhesion and proliferation of Saos-2 cells to the Ti6 Al4V and Ti6 Al4V ac scaffolds were higher on Day 7 and Day 14 compared to Ti6 Al4V ao. The scanning electron microscopy (SEM) images in Figure 12 provide a better understanding of the events that occurred during the in vitro studies. The process of cell attachment involves the formation of filopodia, which consist of microtubules and actin filaments. Filopodia have a finger-like morphology that enables the cell to sense the surface and search for suitable adhesive sites. Thus, a higher number of filopodia indicates favorable compatibility between the cells and the biomaterial surface, as filopodia act as sites for cell anchorage and attachment [33]. Although phyllopodia would have been observed in the cells adhered to the anodized scaffolds, the immunofluorescence images and in vitro studies revealed a low number of adherent cells. Therefore, one hypothesis that can be considered is that the weak adhesion of the cells to the anodized scaffolds led to their detachment during the tests, thereby decreasing the number of cells adhered to the surface. It is important to observe the behavior of the cells that remained adhered to the anodized scaffolds in the ALP tests. In Figure 10, a peak in activities on Day 3 can be observed. This peak is justified by complete exposure to the osteogenic environment, confirming accelerated osteogenic differentiation. This finding is in agreement with studies reported by other researchers [34]. Subsequently, there was a reduction on Day 7, which was very pronounced on Day 14. The detection of calcium in the semi-quantitative analysis with EDS for only the scaffolds in this group (Figure 12e) reinforces the theory that bone differentiation may have contributed to a decrease in the osteogenic environment. However, this hypothesis needs to be supported by additional in vitro and in vivo studies. Figure 11: SEM micrographs of porous Ti6 Al4V scaffolds and cell morphology on porous scaffolds after being cultured on Day 1 for different groups: (**a**) Ti6 Al4V (as-built); (**b**) Ti6 Al4V after chemical polishing (Ti6 Al4V ac); and (**c**) Ti6 Al4V after anodic oxidation (Ti6 Al4V ao); (**d**)Ti6 Al4V after anodic oxidation (Ti6 Al4V ao) image with higher magnification.