## 3 Results and Discussion
After being certain that surfaces are not cytotoxic, it is important to investigate whether they will allow for cell viability, adhesion, and proliferation as they are the most important for osseointegration. A commercially available CellTiter-Blue assay (Promega) was used to assess cell viability after 1, 4, and 7 days of cell culture (Figure 3). Cell viability was determined by calculating the reduction percentage of the reagent from the CellTiter-Blue assay. The reagent solution contains resazurin, which is reduced to resorufin by living cells through dehydrogenase enzymes. Therefore, the higher the expression of resorufin, the higher cell viability [30]. Cells grown on the tissue culture polystyrene well plate were used as a positive control for the assay, and results were normalized with the same. After 1 and 4 days of cell culture, Ti surfaces displayed the highest cell viability, with a slight decrease in PTi surfaces. NPTi surfaces displayed significantly lower viability compared to Ti surfaces. However, after 7 days of culture, all the surfaces displayed equivalent cell viability. \begin{table}
\begin{tabular}{c c c c c} \hline  & **\%Ti 2p3/2** & **\%C 1s** & **\%O 1s** & **\%Na 1s** \\ \hline
**Ti** & 7.38 & 57.43 & 35.20 & - \\
**PTi** & 14.15 & 38.56 & 47.30 & - \\
**NPTi** & 3.62 & 27.53 & 44.78 & 24.07 \\ \hline \end{tabular}
\end{table} 
Table 1: Surface elemental composition for different surfaces [28]. Figure 2: LDH assay results showing the cytotoxicity of surfaces. The adhesion and proliferation of ADSCs were investigated using fluorescence microscopy. After 4 and 7 days of cell culture (Figure 4), as expected, Ti surfaces displayed the most cell adhesion, followed by PTi and NPTi, respectively. NPTi surfaces displayed significantly lower cell counts than both Ti and PTi surfaces. There was a drastic growth of cells on PTi surfaces between 4 and 7 days of culture. However, the cell functionality of the adhered cells was important. The cell growth on Ti surfaces was unidirectional, whereas on PTi and NPTi surfaces, it was more elongated. Not much growth was observed on NPTi surfaces, which also corroborated the quantified results. The lower adhesion could be attributed to the microporosity of the surfaces for PTi and NPTi, with similar studies showing porous surfaces can lead to lower cell proliferation yet higher differentiation [31; 32]. Hence, even though PTi and NPTi surfaces displayed lower adhesion, they have a micro-porous structure that has been established as one that promotes osseointegration. SEM was used to evaluate the morphology of adhered cells (Figure 5). As expected, the cell morphology for Ti surfaces was comparable to that reported in similar studies [33]. As seen with fluorescence microscopy images, an increase was observed in the proliferation of cells from day 4 to 7 of culture. The growth of cells on PTi and NPTi surfaces was elongated, as was seen in the fluorescence microscopy. The elongations are actin-based protrusions called filopodia, and they play a critical role in the cell adhesion process and in sensing their environment for contact guidance [34]. Filopodia are important for the cell response to nanoscale topography [35]. The spreading is also different from Ti surfaces to PTi and NPTi surfaces. Cells are constricted on PTi and NPTi surfaces compared to the spreading seen on Ti surfaces. After 7 days of cell culture, differentiation to osteogenic lineage was induced by providing osteogenic media to cells. The osteogenic media was prepared by supplementing the cell culture growth media, which included \(\beta\)-glycerophosphate, dexamethasone, and ascorbic acid. Supplementing the growth media is necessary as \(\alpha\)-MEM media enhances cell proliferation but disables differentiation markers [36]. Each of the additives has a specific role in influencing the osteogenic differentiation of ADSCs. RUNX2 expression, a key transcription factor for osteoblast differentiation, is induced and regulated by dexamethasone.