# Preparation and Electron-Beam Surface Modification of Novel TiN Material for Medical Applications
## 3 Results and Discussion
### Electrochemical Tests and Cell Growth
#### 3.2.2 Cell Growth  
The preliminary experiment with the model cell growth was conducted on the surface of sample TN. It is seen in Figure 8 that the breast cancer cells partially covered the material surface after a week of cultivation. They can be seen all across the entire surface both as single objects and as small groups. However, the observed surface belongs to the external layer of the material, while the cells possibly grown in the internal layer could not be visualized. To detect the existence of such cells in the hidden region, we compared EIS data obtained in saline for both cell-free samples and those with cells cultivated for 1, 3, and 5 days. Interestingly, the samples populated with cells demonstrated impedance spectra similar to their cell-free counterparts whose typical Nyquist plot was previously shown in Figure 6. Therefore, the same equivalent circuit can be used to describe processes occurring on the surfaces with grown cells. Thus, the presence of growing cells appears not to affect the mechanism of surface processes in saline medium. At the same time, the values of CPE-T and \({\rm R_{p}}\) parameters were found to change over time of cell growth, as shown in Figure 9.  
Figure 8: CLSM image of sample TN after cell growth for one week. Green color is used for visualization of grown MCF-7 cells. The parameters associated with the external layer of the sample (CPE\({}^{1}\)-T and R\({}_{\text{p}}\)\({}^{1}\)) are seen in Figure 9a,b to demonstrate the most dramatic change after as long as just one day of cell growth. Meanwhile, the parameters describing the internal layer (CPE\({}^{2}\)-T and R\({}_{\text{p}}\)\({}^{2}\)) are seen to undergo the most noticeable change between days 1 and 3 of cell cultivation (Figure 9c,d). Since cells are non-conductive biological objects, both layers were expected to increase resistance in their presence. Therefore, the observed drastic increase of parameters R\({}_{\text{p}}\)\({}^{1}\) and R\({}_{\text{p}}\)\({}^{2}\) is a good indication of cell growth on both the external and internal layers of the sample. Since the internal surface was harder to reach for cells, its resistance (R\({}_{\text{p}}\)\({}^{2}\)) is seen in Figure 9 to jump later than R\({}_{\text{p}}\)\({}^{1}\). Note that both CPE-T parameters demonstrated behavior similar to that of their corresponding R\({}_{\text{p}}\) parameters, from which we conclude that the presence of cells somehow resulted in increase of both capacitive components CPE\({}^{1}\)-T and CPE\({}^{2}\)-T.  
As seen in Table 4, time constants \(\tau_{1}\) and \(\tau_{2}\) also demonstrated drastic changes between days 0-1 and 1-3, respectively. The lifetime \(\tau_{1}\) associated with processes in the external layer of the coating increased by 6 orders of magnitude, and \(\tau_{2}\), related to the internal layer, increased by two orders of magnitude. Hence, the presence of cells was found to slow down surface processes. That is why, through the changes of parameters R\({}_{\text{p}}\), CPE and \(\tau\), analysis of EIS data allowed us to observe the appearance and growth of cells on both external and internal layers of the coatings prepared in this study. Eventually, after 5 days of cell cultivation, the CPE-T and R\({}_{\text{p}}\) parameters of both parts of the circuit (as well as parameter \(\tau_{1}\)) somewhat decreased, as well seen in Figure 9. This could be due to possible stabilization of the system TiNi/cells. For deeper understanding, additional and more detailed studies are needed, which was not within the scope of the present work. Thus, using electrochemical techniques it was found that cell growth first begins on the external surface, after which cells start covering the deeper layer of the material by day three of cultivation experiments. Hence, using this relatively simple and easy approach, kinetic studies of cell growth can be realized in the future.