Apart from voltage and anodization time, the preparation of electrolytes affects the growth of TNA, as shown in Fig. 1. Table 1 shows the average diameter of the TNA after anodization in electrolytes aged at different incubation times. The longer the mixing period, the more soluble the fluoride salt, thereby making the release and uptake of ions easy for TNA growth. FESEM analysis of TNA in the electrolyte at 1 and 5 h shows the disorganised formation of TNA compared with TNA anodised in electrolyte aged overnight (16 h).
Table 1
Average diameter of TNA in electrolytes aged at different times at the same anodization period (30 mins).
Electrolyte ageing (h) | Voltage (V) | Average diameter (nm) |
1 | 30 | 66.149 ± 13.6 |
60 | 154.39 ± 11.8 |
5 | 30 | 76.994 ± 9.87 |
60 | 169.53 ± 14.6 |
16 | 30 | 72.517 ± 20.4 |
60 | 121.752 ± 18.5 |
Growth Behaviour And Structural Characteristic Of Tna
The typical current density recorded during anodization of Ti at 30 V in glycerol containing 0.5 wt.% NH4F solution is displayed in Fig. 2. A correlation is observed between the current transient behaviour and changes in surface morphology of the oxide layer formed on Ti during anodization. The current density decreased drastically from 1.8 mA to approximately 1.1 mA within the first few minutes because of the formation of the compact oxide layer (Fig. 3a). The oxidation of Ti was in accordance with Eq. 1.0 (a–d). The electrode reaction was confirmed by the formation of white bubbles surrounding the cathode electrode at the beginning of anodization because of O2 evolution. The formation of the TNA was discussed in detail by Sreekantan et al., [12] and Indira et al. [13].
2H2O + 4H+ O2 + 8H+ (Eq. 1.0a)
4OH− 2H2 + O2 + 4e− (Eq. 1.0b)
Anode: Ti + O2à TiO2 (Eq. 1.0c)
Cathode: 4H+ +4e 2H2 (Eq. 1.0d)
The growth of nanoporous TNA is described in Fig. 3. Random pit formation lasts for 2 min was clearly observed in Figs. 3 (b – c). The interaction of surface Ti4 + ions with oxygen ions in the electrolyte formed an initial oxide layer during the first 10 s of anodization in Fig. 3a, and the compact layer of TiO2 formed on Ti. A large cracked area of porous oxide film and craters at the interface film substrate were observed, which were assumed to be interface spaces amongst the amorphous TiO2 layers that were regarded as the most unstable sites [14], thereby providing easy pathways for ion penetration from electrolyte. A few patches of un-anodised Ti were still present after 1 min of anodization (Fig. 3b), and a complete transformation from Ti to TiO2 porous layer was observed on the sample surface after 2 min of anodization (Fig. 3c). The pores were not self-ordered at this stage, and small pits originated from localised oxide were observed because of dissolution of a chemical process induced by F-ions, where it acted as a pore-forming centre. Hence, the electrolyte ions will easily penetrate into the interface and form pores.
Anodization for 5 min (Fig. 3d) resulted in the separation of individual tubes from the nano-pores to larger pores, where the thickness of the outer oxide layer was approximately 225 nm. As time increases, a distinct nanoporous morphology was observed (Fig. 3e) on the sample surface after 10 min of anodization. The breakdown sites served as seeds to disordered worm-like structure growth (Figs. 3[e–k]). The dissolution of TiO2 from Ti resulted in pH gradient at the top and bottom pore of the tube, where lower pH could accelerate the dissolution and pore penetration into the Ti substrate, thereby resulting in pore transition from irregular to regular formation. Different pores will compete with one another for total available current to form a uniform TNA morphology, and if the current is sufficient, then the pore will survive and retain the morphology of the tube [8]. A small increase in current density approximately 30 min indicated that the nucleation of nanopores and the formation of nanotubular oxide were due to random breakdown of the barrier oxide film on the Ti surface and dissolution of F− ions. Dissolution of TiO2 to form pores is performed according to Eq. 1.1.
TiO2 + 12F− + 4H+ TiF62− + 2H2O (Eq. 1.1)
A sample anodised for 20 min (Fig. 3f) has a better appearance because of the less debris on the anodised surface compared with the sample anodised for 10 min. Thus, the transformation of pit to a tubular structure occurred during 5 min to 20 min of anodization. However, compact oxide partially covered the surface. When anodization reached 30 min (Fig. 3g), the oxide layer was completely dissolved, and a self-ordered TNA structure could be observed. A similar morphology to the sample anodised for 30 min can be seen with the remaining samples of Ti anodised for 1, 2 and 3 h (Figs. 3[h–j]). The inner diameters of TNA remained constant with time, but the length increased with the increase of anodization time beyond 30 min [15]. At this stage, the predominant formation of the oxide layer was maintained, resulting in the formation of a thick oxide layer with slow chemical dissolution. As the experiment continued, chemical dissolution actively proceeded according to Eq. 1.1. After 6 h of anodization (Fig. 3k), TNA started to collapse and peel off from the Ti despite the increase of TNA length. Therefore, for a well-developed TNA, the anodization time should be within 30 mins to 3 h.
Forming a nanoporous structure has two basic mechanisms based on the result shown in Fig. 1 and Fig. 3: (1) electrochemical oxidation and (2) chemical dissolution. Under the application of electric current, the initial TiO2 layer or barrier layer was formed on the metal surface, and pore initiation occurred because of electric field-assisted dissolution with the presence of fluoride (F-) ions at the preferred sites of the oxide layer. These oxidation and chemical dissolutions were active at the bottom of the pore. The barrier layer was reduced by pore formation, whereas concentrated electric field intensity beneath the barrier layer ensured further growth of the pore [16].
The TNA growth rate has three different regions (Fig. 4). At stage I, the electric field intensity increased because of the accelerated dissolution of TiO2 at the bottom of the pits, thereby making the barrier layer at the bottom relatively thin and resulting in further pore growth [17]. On the contrary, larger tubes were produced by electrochemical etching of F-ions. The formations of TNA become slower during the second stage because the size of inner diameter of TNA slightly changed, and less F− ions produced. Furthermore, in stage II, when the rate of chemical dissolution and electrochemical etching was at equilibrium, the growth rate of TNA formation become constant throughout the process. During the final stage, electrochemical etching was slower than that at stage II, thereby hardly making any changes on the TNA inner diameter because of the absence of F-ions. Hence, the rate of TNA length growth increased because of the suppression of chemical dissolution towards electrochemical etching.
Cell Morphology Of Tna
In the present study using FESEM, the response of different cells seeded on glass, Ti and TNA were observed to identify the appropriate surface modification for the characterisation of biomaterials. A similar morphology to cell adhesion was visualised under FESEM by different types of human cells. Cancer cell lines were mostly studied because of the rapid growth of the cells compared with normal cells [18]. In this study, several cancer cells were studied on the cell attachment profile, such as (a–c) SH-SY5, (d–f) NPC/HK-1, (g–i) C666-1, (j–l) HT 29, (m–o) HEK 293 and (p) SaOS-2. The initial stages of cell adhesion were critical for biomaterial integration. After optimizing the ideal anodization time of TNA, 30 min at 30 V with a sweep rate of 1 V/s was selected for investigating cell integration due to the clear, well-aligned TNA produced. FESEM analysis of different types of adherence cells showed the growth pattern of cell adhesion on the TNA surface after 48 h of cell culture (Fig. 5).
The FESEM image of the human neuroblastoma SHSY-5Y cell line revealed that the cellular protrusion adhered to the TNA surface, as shown by the yellow arrows in Figs. 5 (a–b). Interactions between filopodia (Figs. 5 [a–p]) supported the properties of TNA to stimulate cell proliferation [19]. The SaOS-2 osteoblast cells spread and proliferate to one another with the presence of small calcified nodules observed in Fig. 5 (p). Moreover, the cells showed a three-dimensional network-like structure (Figs. 5 [d, g, l, n and p]). Cells tended to elongate with many filopodia interacting with one another via cellular extension from the cell to the substrate. The extension of cells on the TNA was stronger and wider compared with those on glass and Ti surfaces. A report has mentioned the importance of the TNA nanosurface with rough topography in promoting cell attachment [20]. The cell network for fibre-like cell spreading on top of the TNA surface makes anchoring more stable, particularly after 48 h incubation [21]. The diameter of the TNA, ranging from 15 nm to 100 nm, played an important key role in supporting cell adhesion [22].