Figure 1 Fabrication of nanotubes via a porous hard template such as AAO.

Figure 2 Electrochemical etching anodization set-up for the synthesis of TNT. Reproduced by permission of The Royal Society of Chemistry [41].

Figure 3 Schematic illustration of TiO2 nanotube formation [46]. Reproduced from Jin et al.

Figure 4 Human osteoblast cell attaches to nanotubular surface while filopodia penetrates into porosities as anchorage sites [10]. Reproduced with permission from John Wiley and Sons Inc.

Figure 5 Clustering of integrins, formation of focal adhesions, MSCs spreading, actin polymerization, and osteogenic differentiation are increased on a nanotubular surface with 15 nm lateral spacing, in presence of BMP-2 signaling [59]. Reproduced with permission from John Wiley and Sons Inc.

Figure 6 Schematic illustration of TiO2 nanotube formation [46]. Reproduced from Jin et al.

Figure 7 Staining actin cytoskeleton of MC3T3-E1osteoblast on: (a) smooth surface, (b) non-annealed nanotubular surface, nanotubular surfaces annealed at (c) 450 °C and (d) 550 °C. Compared with the smooth and the non-annealed nanotubular surfaces, annealed surfaces show higher regular arrangement [9]. Reproduced with permission from John Wiley and Sons Inc.

Figure 8 SEM images of TNT fabricated during varying durations [88].

Figure 9 Intercalation of drug inside the nanotubes. Reproduced by permission of The Royal Society of Chemistry [41].

Anodization Condition

Annealing
Conditions

Cell Type

Cell Culture Duration

Effect of TNT Size

Ref

Anode:Titanium foil
Cathode: Platinum
Electrolyte: 1M H3PO4 and 0.5 wt % HF
Duration: 3h
Voltage: 5 to 25 V

 

450 °C for 3 h

MC3T3-E1

2h before Cell adhesion. 24, 48, and 96h before MTT. 1, 2, and 3 weeks for ALP activity. 3 week before Alizarin R-staining.

Diameter of 20–70 nm enhanced cell adhesion,
alkaline phosphatase activity, and mineralization. The proliferation increased with increasingtube diameter from 20 to 120 nm.

[70]

Anode:Titanium foil
Cathode: Platinum
Electrolyte: 1:7 volumetric ratio of acetic
acid to 0.5% w/v hydrofluoricacid in water
Duration: 30 min
Voltage: 5, 10, 15 and 20 V

500 °C for 2 h

MC3T3-E1

2h, 12h, 24h, 48h, 72h and 7d for cell counting. 24h and 48h for MTT and ALP.

30 nmnanotubes enhanced osteoblast adhesion, while 70–100 nm nanotubes provide a lower population
of cells with elongated cellular morphology and enhanced alkaline phosphatase levels.

[34]

Anode: Titanium sheet
Cathode: Platinum
Electrolyte: 0.5 wt % hydrofluoric acid and acetic acid volumetric ratio  7:1acid in water
Duration: 30 min
Voltage: 5, 10, 15 and 20 V

500 °C for 2 h

hMSCs

2h, 48h for Cell adhesion. 3 weeks for osteogenetic markers microscopy.

30nm diameter nanotubes enhanced adhesion, while
70to 100 nm diameter nanotubes show stem cell elongation and selective differentiation into osteoblast-like cells.

[56]

Anode: Titanium sheet
Cathode: Platinum
Electrolyte: 1 M H3PO4with addition of 0.3 wt%HF
Duration: 1h
Voltage: 1 V up to 20 V

 

 

hematopoietic stem cells (HSCs), human osteoblast-like

2 weeks
with differentiation medium before analysis by Immunocytochemistry.  3 days before osteoblast cell counting.

Diameters between 15 and 100nm were verified. 15nm supports HSCs differentiation into osteoclasts, adhesion and osteoblast proliferation.

[57]

Anode: Titaniumfoil
Cathode: Platinum
Electrolyte: 1 M H3PO4with addition of 0.3 wt%HF
Voltage: 1 V up to 20 V

 

Rat mesenchymal stem cells

2weeks before analysis by immunocytochemistry. 3 and 6 days before cell counting.

Diameter less than 30 nm with a maximum at 15 nm enhanced integrin clustering/focal contact
formation and cellular activities.

[49]

Anode: Zirconium and titanium foils
Cathode: Platinum
Electrolyte: For Zr, 1 M (NH4)2SO4 with theaddition of 0.15 M NH4F. For Ti, 1 M H3PO4 with the addition of 0.125 M HF

 

Rat mesenchymal stem cells

1 day before cell adhesion and 3 days before cell proliferation.

Both materials provide enhanced cell adhesion and proliferationwith nanotube diameters of 15–30 nm.

[58]

Anode: Titanium foils
Cathode: platinum
Electrolyte: 1 m H3PO4 with addition of 0.12 M HF
Voltage: 1 and 20 V

 

mesenchymal stem cells

24h for cell counting. 2 weeks in differentiation medium before immunocytochemistry.

Differentiation is enhanced on 15 nm but not on 100 nmBMP-2-coated nanotubes.

[59]

Table1 Effect of nanotube diameter on cellular behavior

Time
V

4h
(mm)

8h
(mm)

16h
(mm)

20 V

0.589

1.07

1.39

40 V

1.443

4.53

6.11

60 V

5.493

6.75

10.08

Table2 Effect of anodization time and voltage on TNT length.