## 3 Results
### XRD Analysis
Previously, a complete report on ATZ patterning was done and demonstrated selectively structured morphologies with outstanding topographical properties [48]. On all surfaces, viable cells were detected (green) (Figures 9 and 10). When compared with the positive control (Figure 10), where cells were seeded on the tissue culture polystyrene, fewer cells were present on the samples. This was also confirmed by the Alamar Blue assay (Figure 11), where the fluorescent signal for the positive control was much higher in comparison with the signal from the samples. After a culture period of 7 days, the cells start to elongate, following the pattern on the scaffold (white lines) (Figures 9 and 10). Detailed images without the red filter show the clear cell elongation (Figure 9). An Alamar Blue Assay was performed on days 1, 4, and 6 to evaluate cell proliferation. Cells showed a limited proliferation in the function of time. Moreover, for some samples (\(\beta\)-TCP S5 and ATZ 6), there is a decrease in proliferation. In general, cell proliferation was the highest on the B-TCP S11 and ATZ 5 samples. This was also confirmed by live/dead assay (Figures 9 and 10), where more viable cells were seen on both samples. However, the tests performed with Alamar Blue Assay did not show the highest signals for cell proliferation, but there is a sign showing different sensitivity in close relation to the type of patterning performed. The cells were monitored on different patterned substrates at 1, 4, and 6 days. As seen from the graphs, cells were able to grow in different levels of the scaffold. As a basic tendency, a lower fluorescence signal was determined for the samples treated in the type one regime (Table 1)--the continuous scanning with two diverse velocities and in the cases of pulse selection regime (type II) with low scanning speeds. The results from the Alamar Blue(r) Assay are given in the form of a graph (Figure 11)--the five different samples of \(\beta\)-TCP pellets and two ATZ samples treated with different parameters of the fs laser (as given previously in Table 1) were fluorescently monitored on the first, fourth, and sixth day after cell seeding according to a positive and negative control. As expected from these first preliminary cellular results, the different parameters of the fs laser patterning influence MC3T3 cell growth--for example, the highest fluorescent signal is observed in the case of \(\beta\)-TCP S11, where spot by spot areal scanning by the laser was performed (N = 1, 2, 5, 10; F = 0.61 J/cm\({}^{2}\)). With increasing fluence (\(\beta\)-TCP S10 and S12), at the same scanning mode of the laser, the fluorescent signal decreases for day 1, day 4, and day 6. The results obtained \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Sample \(\mathcal{N}\)**} & \multirow{2}{*}{**\(\boldsymbol{\
u}\) (KHz)**} & **Type I Regime:** & **Type II Regime:** & \\  & & **Continuous Scanning.** & **Spot by Spot Scanning** & **F (J/cm\({}^{2}\))** \\  & & **(Scan Speed (mm/s))** & **(Number of Laser Pulses (N))** & \\ \hline
1. \(\beta\)-TCP S5 & 500 & 1. 7; 3.8 & - & 1.03 \\
2. \(\beta\)-TCP S6 & 500 & 1. 7; 3.8 & - & 0.61 \\
3. \(\beta\)-TCP S10 & 25 & - & 1, 2, 5, 10 & 1.03 \\
4. \(\beta\)-TCP S11 & 25 & - & 1, 2, 5, 10 & 0.61 \\
5. \(\beta\)-TCP S12 & 25 & - & 1, 2, 5, 10 & 1.86 \\
6. ATZ 5 & 500 & 1.7; 3.8 & - & 1.86 \\
7. ATZ 6 & 500 & 0.5; 0.8; 16 & - & 1.86 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Parameters employed for texturing in two different regimes of femtosecond laser patterning. in the case of continuous scanning regime are analogical, where the higher fluorescent signal is again observed at the lower fluence applied (\(\beta\)-TCP S6). Another interesting observation is the behavior of seeded ATZ samples.