*3.2. Surface Morphologies of Nano and Micro/Nano-Structures of Al Template*

To assess the quality of the prepared anodized aluminum templates, they were observed using SEM and AFM, as presented in Figure 3. Figure 3a shows the SEM images of AAO that were formed by the anodization process. The results reveal that the mean pore size in AAO was approximately 100 nm. Additionally, the formed pore arrays of AAO were very uniform. The micro/nano-structure of the Al template obtained by micro-powder blasting and anodic oxidation process is discussed. Figure 3b presents an SEM image for the previous process with electrolytic polishing, followed by the anodic oxidation process. The results demonstrate that micro-powder blasting barely formed a micro-structure, but rather formed nanoholes in AAO, yielding a pore size of about 60–80 nm. Figure 3c shows the SEM images following anodic oxidation process without electrolytic polishing. The results indicate that the micro-structure formed on the Al template, and nanoholes formed in the micro-structure with sizes about 50–80 nm, suggesting that the micro/nano-structure formed on the Al template.

**Figure 3.** SEM images of nano-structure and micro/nano-structure: (**a**) Φ = 100 nm (anodic aluminum oxide (AAO)); (**b**) Φ = 60–80 nm (nano-hole on micro/nano-structure with electrolysis polishing); (**c**) Φ = 50–80 nm (nano-hole on micro/nano-structure, without electrolysis polishing).

#### *3.3. Surface Properties of Various Al Template Structures*

The surface properties of the various structured templates importantly affect the cell culture thereon. The effects of contact angle and surface roughness of Al templates with various structures on their surface are considered. Table 1 displays measured contact angles on smooth Al template (flat-structure), a micro-structured Al template that was formed by micro-powder blasting (MS 245A, MS 300A and MS 550BT), the nano-structure (AAO) and the micro/nano-structured Al template, revealing that the contact angles of the Al templates with the different structures fall into three groups. The contact angles of the smooth Al template and the micro-structure of Al substrate formed by micro-powder blasting (MS 245A) were about 77◦–88◦. The contact angle of Al template by MS 245A did not decline very much as the size of the sand particles increased, yielding a larger micro-structure, so the surface properties of the Al template did not improve with an increase in the particle size. The contact angles of the Al template with micro-structure formed by micro-powder blasting (MS 300A, MS 550BT) were around 28◦–36◦, revealing that the surface of the Al template by micro-powder blasting changed from hydrophilic to more hydrophilic. Furthermore, the contact angles of Al templates with various structures were affected by the size of sand particles, and are independent of their shapes. Finally, the contact angles of the nano-structure (AAO) and micro/nano-structure on Al template were about 7◦–21◦, indicating that these structures are superhydrophilic. These results also show that the micro/nano-structured Al template had the lowest contact angles, and that the micro/nano-structure of the Al template was more hydrophilic than the other structures of the Al template. Contact angle values by Tukey-test are also listed in Table 1. The contact angle indicates that there was no statistically significant difference between the smooth Al template and the Al template with micro-structure (MS 245A). The results also show that the contact angle between Al template with micro-structure (MS 300A) and the Al template with micro-structure (MS 500BT) had no statistically significant difference. The other two had statistically significant differences from each other in terms of contact angle for different structured templates.

Table 1 also presents the surface average roughness (*R*a) values of Al templates with various structures. The results of surface roughness (*R*a) indicate that the *R*<sup>a</sup> of the Al template after micro-powder blasting is larger than that of flat Al template. The Wenzel equation appears that the surface roughness of the template can improve the surface wetting property. The contact angle of the template decreased as its surface roughness increased [34]. The results indicate that micro-powder blasting with smaller sand particle yielded larger *R*<sup>a</sup> values of the Al template, because larger sand eroded the Al template more strongly and it could not produce small bumps on the surface of the Al template. The *R*<sup>a</sup> value of the Al template fell as the size of the sand in the micro-powder blasting increased. The results also demonstrated that rounder sand in micro-powder blasting yielded smaller *R*a values of the Al template, because sharp sand could more easy produce bumps on the surface of the Al template. The micro/nano-structure of the Al template had the highest *R*<sup>a</sup> value. The Tukey-test for surface roughness of Al templates with various structures is also listed on Table 1, indicating that there was no statistically significant difference between the nano-structured (AAO) template and the micro-structured template (MS 245A). The results also reveal that the surface roughness between the micro-structured template (MS 300A) and the micro/nano-structured template had no statistically significant difference. The other two had statistically significant differences with each other in surface roughness for different structured templates.

The results of this study also reveal that the micro-powder blasting + anodized method yielded the minimum contact angle and the maximum surface roughness in the Al template. The contact angle had a smaller value and the surface roughness had the smallest value via the anodized method. The contact angle was largest via micro-powder blasting with different sized particles.


**Table 1.** Contact angles and surface roughnesses for different structured templates by Tukey-test.
