## 4 Discussion  
The results of this study showed that CTO 800, a CaTiO\({}_{3}\) film prepared with a heating temperature of 800 \({}^{\circ}\)C, had a highly hydrophilic surface. The ST-2 cells cultured on substrates and CTO 800 exhibited increased cell differentiation, represented by ALP activity, compared with that of Ti and CTO 700. The CTO 800 showed a push-in test value, which indicates the degree of osseointegration, that was significantly higher than the corresponding value for non-coated Ti. The sessile drop method is commonly used because it easily measures the wettability of implant surfaces. The surface wettability of a biomaterial has been reflected by its topography, surface energy, and surface charge. Hydrophilic surfaces have been reported to accelerate the initial interactions between the surface and the wetting liquid, which is important for wound healing and osseointegration [16]. In this study, surface topographies were determined by both SEM observation and EDX examination; three-dimensional surface images were obtained and surface roughness was measured by a three-dimensional non-contact tester. [PERSON] and [PERSON] [17] classified implant surface into four categories and reported that a moderately rough surface (surface roughness, Sa = 1-2 \(\upmu\)m) might have advantages over other surfaces. [PERSON] [18] reported that a moderately rough surface (Sa = 1-2 \(\upmu\)m) had a considerable impact on osteoblast differentiation. Furthermore, hybrid micro/nanostructure implant surfaces have been reported to affect osseointegration through managing cell behaviors; micro-nano hybrid structure surfaces increased  
Figure 6: Push-in test values for Ti and CTO 800. Data are mean \(\pm\) standard deviation (\(n\) = 5). the wettability of such implants [19; 20; 21]. In this study, the Sa values for cpTi, CTO 700, and CTO 800 were relatively smoother than those of the prior reports (0.49 \(\upmu\)m, 0.62 \(\upmu\)m, and 0.87 \(\upmu\)m, respectively). However, there were significant differences of Sa between Ti and CTO 800, CTO 700, and CTO 800. In addition, the surface morphology of CTO differed depending on the heating temperature; CTO 800 showed a cauliflower-like texture comprising nanoparticle structures (Figure 2F). This unique surface morphology might affect wettability and lead to accelerated cell differentiation; however, it did not impact proliferation. Most studies have reported that hydrophilic surfaces have the potential to enhance the early stages of cell adhesion, proliferation, differentiation, and bone mineralization compared to hydrophobic surfaces [22; 23]. On the other hand, opposite results have been found in studies using different surface energy which cause by UV exposure times, and it is possible that extremely high surface energies promote cell adhesion but suppress the spreading and differentiation of cells. The cell numbers on superhydrophobic surface were lower than those on hydrophobic surfaces materials for 24 h incubation; however, the ALP activity of superhydrophilic surface was significantly increased compared to the others [24; 25; 26]. Osteoblast differentiation can be characterized in three stages: cell proliferation, matrix maturation, and matrix mineralization. The matrix maturation phase is characterized by maximal expression of ALP, an outer cytoplasmic membrane-localized enzyme for phosphate ester hydrolysis. Therefore, ALP is one of the most commonly used markers for osteoblastic cell differentiation at an early stage. Alkaline phosphatase activity is upregulation after proliferation begins to decline and genes for proteins such as osteoclast and osteopontin are expressed, finally, mineralization is completed [27; 28; 29]. In this study, the cell might be proliferated to a confluent state within 7-14 day observation periods. As expected, the high ALP expression of CTO 700 and CTO 800 was observed at day 7 and followed by downregulation of ALP expression at day 14. On the other hand, the level of ALP activity of Ti was not changed dramatically.