A Review on TiO2 Nanotubes: Influence of Anodization Parameters, Formation Mechanism, Properties, Corrosion Behavior, and Biomedical Applications
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In this article, influence of anodization parameters on the formation of tubes, tube dimensions, formation mechanism, properties of TiO2 nanotubes (TNT), and their applications in biomedical field are reviewed. The fabrication of TNT of a different shape such as pore size, length, and wall thickness by varying anodization parameters including electrolytes, pH, voltage, electrolyte bath temperature, and current density is examined and discussed. The crystallographic nature of the nanotube obtained by various methods has also been discussed. Finally, the article concludes by examining the key properties including the corrosion aspect and various applications in biomedical field in depth.
KeywordsTiO2 nanotubes Oxide dissolution Amorphous Corrosion Biomedical applications
Now-a-days, a wide range of materials are required to design and study modern devices, which are suitable for various possible commercial applications. Nanomaterials play an important role in recent technologies to reach high-performance devices. The performance of such devices is significantly determined by geometry, shape, and morphology of nanostructures . Exponential growth in the literature addresses that the nanoscale began at 1990’s . Interest in the nanoscale has been driven by the commercial availability of nanoscale manipulation and characterization tools due to the following reasons: (1) expectation of new physical, chemical, and biological properties of nanostructures, (2) expectations that the nanostructure provides new building blocks for innovative new materials with novel properties, (3) miniaturization into the nanoscale by the semiconductor industry, and (4) recognition that the molecular machinery in a biological cell functions at the nanoscale . Historical aspects of chemistry and biology such as colloids, protein engineering, and molecular virology have involved nanostructures but on a largely empirical basis. Finally, there is an expectation that a better understanding of the 1–100 nm size materials (nanoscale) leads to a seamless integration of theory and models across the size scales that encompass atomic–molecular nanostructure–microstructure behavior, and thereby enable the priori prediction and design of a material’s properties .
1.1 Various Methods for the Development of TNT
Numerous methods are available for the development of TNT, which includes anodization [55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65], sol–gel method [66, 67, 68, 69], template method [70, 71, 72], hydrothermal method [38, 73, 74, 75, 76, 77, 78, 79, 80, 81], sonoelectrochemical [82, 83, 84, 85], micro-wave irradiation [86, 87], and alkaline synthesis . Hoyer  was first reported the synthesis of TiO2-based nanotubes via template-assisted synthesis. Thereafter, electrochemical anodization and hydrothermal methods were succeeded in fabricating TNT .
1.2 Nanotube Arrays on Ti Alloys
Nanotubes can also be fabricated using Ti alloys as substrate materials. However, most of the Ti alloys used in engineering/biomedical applications show a dual phase that is α + β microstructure. The α phase, which has a hexagonal close packing (hcp) structure, is enriched with hcp stabilizing elements such as Al, O, N etc., whereas β-phase with body-centered cubic (bcc) structure is enriched with the β-stabilizing elements such as V, Nb, Mo, Ta, etc. The formation of uniform nanotubular oxide layer on Ti alloys having dual phase microstructure cannot be achieved which is due to the difference in chemistries of these phases, as one phase could get etched preferently by the electrolyte. It was envisaged that the differences in chemistries and dimensions of nanotube on Ti alloys could render interesting electronic and physical properties that can be modulated for a wide variety of applications . The mainly used Ti alloys for the formation of nanotubes are Ti–Nb , Ti–Ta , Ti–Al–Zr , Ti–Zr , Ti–29Nb–13Ta–4.6Zr , Ti–Mn , Ti–6Al–4V , and Ti–6Al–7Nb .
1.3 Doping of TNT with Other Elements
Most recent research on TNT focuses on the doping or deposition of some metal ions such as Ta , Au [33, 68, 101], Ag [102, 103, 104], Ru , B , Fe , Cu , Pd , Pt [109, 110], Ni [111, 112], Cr , Nd , Sr [115, 116], Zr [117, 118]; and non-metals like C [20, 119, 120, 121, 122], N [123, 124], and F and B ; and metal oxides like MnO2  had enhanced its applications in many fields. Most recently, Loget et al.  coated biomimetic polydopamine into TNT for the development of biomedical devices. Similarly, graphene oxide coating has been done on TNT for biomedical applications . The TNT can be used as a field emission electron source for generating X-ray radiation. It is not affected by exposure to O2 and has a good electrical contact between TNT film and conductive Ti sheet .
2 Influences of Anodization Parameters
Great attention has been drawn on the formation of nanotubes which were known to be affected by the electrolyte concentration, electrolyte pH, anodization voltage, time, and temperature . Therefore, the following section provides the comprehensive review on the influences of aforementioned parameters on the formation of TNT.
2.1 Effect of Electrolytes
The nature of electrolyte used for the development of TNT strongly influences the formation of the graded structure. It was well recognized that under the same conditions, different electrolytes may produce different electric field intensities. It was also well known that in the initial stage of anodization, the higher electric field intensity can induce bigger breakdown sites which finally result in wider diameter of TNT. Additionally, the chemical dissolution rate of oxide layer is also discrepant in different electrolytes. In other words, one can obtain different titanium oxide structures such as a flat compact oxide, a disordered porous layer, a highly self-organized porous layer, and/or finally a highly self-organized nanotubular layer by controlling the electrochemical anodization parameters of Ti .
The TNT can be synthesized by anodization using various fluoride-containing electrolytes like NH4F/CH3COOH, H2SO4/HF, Na2HPO4/NaF, etc. The nanotube diameter is remarkably affected by the electrolytes used . Generally, two different types of electrolytes such as organic (or neutral) and aqueous electrolytes have been used for the synthesis of TNT. When the anodization takes place in an acidic condition of pH, in other words, high water content is called aqueous electrolytes. An organic (or neutral) electrolyte has a small amount of oxygen/water content in comparison to an aqueous electrolyte.
In the organic electrolyte like (NH4)H2PO4/NH4F  and (NH4)2SO4/NH4F , longer nanotube length and better self-organized TNT layers were obtained. These self-organized porous structures have large potential and commercial applications in view of several interesting properties. Yang et al.  reported that in order to obtain long TNT, fluoride-containing electrolytes should be used in place of HF-containing buffer electrolytes species of highly viscous (organic) electrolytes. Neutral and viscous electrolytes allow better control in the anodic conditions and creating two different environments along the tube, one inert at the top of it and other chemically reactive at the bottom. Thus, the inhibition of the oxide dissolution at the top side of the nanotube allows the formation of longer TNT . Much higher current densities are observed in the aqueous electrolytes than in the organic electrolyte , which is attributed to higher diffusibility and concentration of ions in aqueous electrolytes. However, smooth curves were obtained at all voltages in the glycerol (organic) electrolytes, whereas current fluctuations were observed in aqueous electrolyte which is due to the high rate of chemical dissolution and the oxidation of Ti .
Furthermore, it is also evident that the layer in the acidic electrolytes is relatively thin compared to neutral electrolyte. This difference must be ascribed to pH dependence of the oxide dissolution rate . Aqueous electrolytes like H3PO4/HF , HF/NH4F  etc., provide well-ordered TNT. According to Chen et al. , smaller TNT was obtained using CH3COOH/NH4F electrolytes and can be replaceable by the environment benign electrolytes such as HCl/NH4F electrolytes. Highly ordered TNT was obtained in malonic acid solution . Similarly, Tian et al.  reported that TNT formed in H2SO4 electrolyte exhibited a variation in the tube length, which depends on its concentration; the inner diameter of nanotubes decreases with H2SO4 concentration from 120 to 100 nm in 0.24 mol/l M H2SO4 and 1.75 mol/l M H2SO4, respectively. The difference in concentration leads to significant variations of the structure of Ti. The sponge-like feature was observed at 0.10 wt% NH4F, and at 0.50 wt% NH4F, nanotubular structure is clearly visible. The NH4F concentration is beneficial to the growth of TNT to a certain extend . In addition, TNTs were developed in fluoride-free electrolytes too. Hahn et al.  had successfully developed high-aspect ratio TNTs using fluoride-free approach i.e., the anodization in perchlorate and chloride-containing electrolytes. The ultrahigh-aspect ratio TNT was fabricated by anodization in electrolytes containing oxalic, formic, or sulfuric acid and chlorine ions. Chlorine-based (fluoride-free) electrolytes have an advantage over fluoride containing one. In the fluoride-free electrolyte, long TNT can be grown in a very short time (growth rate is >1000 times faster than in fluorine media; e.g., 10 min in chlorine-based electrolytes compared to ∼17 h in fluorine-based electrolytes) . Nguyen et al.  were developed TNT in fluoride-free electrolytes with bromide anions in various solvents, including ethylene glycol, glycerol, and water–ethylene glycol and water–glycerol mixtures. They found that tubes grown in Br-based electrolyte do not have the self-organized structure of those seen in fluoride solutions, the growth rates are much faster, achieving tens of microns in length in 60s.
Further, reusing the electrolyte solution, which was electrolyzed by previous anodization run, resulted in a better nanotubular morphology than the morphology observed using fresh electrolyte solution . Compared to that observed for the freshly prepared electrolyte, reusing the electrolyte as the second time was found to give the larger pore diameter and the smaller wall thickness, which is attributed to the pronounced effect of more chemical etching or more oxide dissolution influenced by the improved electrolyte conductivity. Normally, after anodization, the field-assisted dissolution led to the dissolution of more titanium ions into the electrolyte; thus, it gives rise to the high electrolyte conductivity. This could in turn fasten the reaction processes in the second anodization and consequently influence the nanotube morphologies. Affected by the electrolyte properties, the increased conductivity of the used electrolyte is believed to play a major role in governing the nanotube formation, and thus determining the consequent film properties .
2.2 Effect of Electrolyte pH
The pH of the electrolyte is the key to achieve growth of the high-aspect ratio nanotubes and it can affect the self-organization behavior of TNT. The difference in the pH leads to significant variations in the pore diameter. The differences in thickness are attributed to pH dependence of the oxide dissolution rate, i.e., dissolution rate at low pH is much greater than the higher pH . Therefore, the pH alters the thickness and diameter of the pores. It has been reported that a mixture of 1 M Sodium hydrogen sulfate monohydrate and 0.1 M KF with 0.2 M Sodium citrate tribasic dehydrate electrolyte shows the variation in the nanotube length from 380 nm to 6 µm and tube diameters from 30 to 110 nm, which is due to the electrolyte pH and anodization potential . The pH value of the electrolyte changes during anodization because of several other reasons . The formation of hydrolysis product at the working electrode leads to the decreases in pH; thus, the local acidification is established. Tailoring the local acidification could promote the TiO2 dissolution, thereby providing more protective environment against the dissolution along the tube mouth, thus, longer tubes were obtained. Hydrogen evolution and OH− species formation take place at the counter electrode [153, 154].
2.3 Effect of Applied Voltage
The applied voltage strongly influences the pore diameter, inter pore distance, and film thickness in a wide range . Subsequently, the morphology of the layer is strongly affected by the applied voltage and anodization time. At low applied voltages, tubes with few hundreds of nanometer in length and a few tens of nanometer in diameter were formed. In the short-term anodization, the individual tubes are connected with each other via rings on the side walls of the tubes, whereas in the longer-term anodization, tubes were separated due to the dissolution of these rings .
The most regular porous structure is obtained at the applied voltage of 20 V. The films formed at potentials below 20 V exhibit a nanoporous but thinner structure. In this case, the layers do not thicken more, because the current is not sufficiently high. It has been reported that the films formed between 22 and 30 V have tubular but irregular porous structure. Above 30 V, large cracked areas of porous oxide film were formed at the interface film substrate with a diameter in the micrometer scale, which can be attributed to breakdown events at high potentials . At low anodizing voltages, the morphology of the porous film is similar to that of porous alumina. As the voltage was increased, the surface becomes particulate or nodular in nature. As the voltage was further increased, the particulate appearance is lost and discrete, hollow, cylindrical tube-like features was created. The structure of nanotube is lost at anodizing voltage greater than 40 V, and a sponge like randomly porous structure was formed .
The chemical dissolution of TiO2 layer plays a crucial role in the TNT formation. As we already seen in the previous section, the chemical dissolution rate of TiO2 is obviously a highly pH-dependent process [151, 156]. Since high acidic condition was established at the bottom of tubes due to the formation of hexafluoro titanium complex (detailed mechanism is given below in the following section), tubes would preferentially continue to grow down to the bottom of metal foil, resulting in tubular structures. Subsequently, high potential will impose more force on the dissolution of oxide by F-ions, the pore size of the TNT will be therefore larger and tube length longer at high anodizing potentials when organic electrolytes were employed . Generally, the dissolution rate is high in the acidic electrolytes due to the higher pH (local acidification) at the bottom of tubes , if more potential is applied in addition to these results in too much dissolution rate, thus it led to permanent dissolution of oxide layer and no tube formation could be observed. Hence, it is preferential to apply lower voltages for the acidic electrolytes and higher voltages for the organic electrolytes.
2.4 Effect of Electrolyte Temperature
2.5 Effect of Current Density
2.6 Effect of Anodization Time
The anodization time also influences greatly on the TNT formation mechanism. There has no TNT, if the anodization time is too short. According to Li et al. report , the minimum time needed for the formation of TNT is 15 min, with increase in time highly ordered TNT can be formed. Similarly, Regonini and Clemens  found that at the anodization time of 5 min, only nanoporous structure was formed, and only after 10–20 min, the nanotubular structure was visible clearly. A short and narrow TNT can be easily obtained in short growth time (i.e., 5-min anodization time) . A report by So et al.  showed that highly ordered TNT with the thickness of 7 µm can be grown in 25 s using Lactic Acid as an additive to the electrolyte. They showed that Lactic Acid can effectively prevent localized dielectric breakdown of the anodic oxide at elevated anodization voltages; thus, it allows establishing a higher ion transport through the oxide film and, as a result, extremely fast tube growth rates without losing the tube’s functional properties. By extending the anodization time, the length of TNT has been increased. If the anodization time goes beyond 1 h, the tubes were collapsed partially due to the over dissolution of the tubes at the top by fluoride ions [163, 164].
2.7 Effect of Electrodes
Apart from the nature of electrodes, the distance between working electrode (WE) and CE also influences the morphology of TNT. How the distance between WE and CE affects the change in electrolyte properties and corresponding morphological features of TNT is experimented by Yoriya . Enlargement of pore diameters, wall thickness, and inter-tubular spacing could be achieved by simply reducing the inter-electrode spacing under a fixed condition. The electrolyte conductivity and titanium concentration were found to strongly depend upon the electrode spacing, with the closer electrode spacings reflecting high conductivity and high titanium concentration. Electrolyte conductivity and titanium concentration are found to drastically increase with decreasing anode–cathode separation. Resulting TNT also tends to increase significantly, particularly observed in inter-tubular spacing as reducing the electrode spacing from 4.5 to 0.5 cm under a fixed electrolyte condition. Due to the combination effect of electrolyte properties and high field strength between the electrodes, the self-enlargement potential is believed to be a driving force for nanotube separation. The unique characteristic of discrete, well-separated nanotube structure is expected to extend and enhance the applications of TNT. While for the case where the CE is far removed from the WE, a significant decrease in pore size and a large increase in number of TNT are obtained. The apparent decrease in pore diameter is probably due to the significant IR drop along the current path in the electrolyte reducing the field strength at the anode electrode. The growth rate of the TNT decreases with increase in the electrode distance.
3 Formation Mechanism of TNT
Since the initial small pores are very narrow, a mass of H+ ions are generated at the bottom of the pore, where the Ti came out of the metal and dissolved in the solution and the concentration of HF inside the pores increases rapidly. High concentration of HF makes the wall of pores dissolves until the adjacent pore walls disappear. At the same time, the integration by small pores resulted in the appearance of larger pores . Simultaneously, the enhanced electric field-assisted dissolution at the pore bottom leads to deepening of the pore and the voids starts forming. Due to the applied electric field, the Ti–O bond undergoes polarization and weakened which promotes the dissolution of the metal oxide .
The formation of ordered film is mainly dictated by the potential distribution at the Ti surface. Two adjacent pores tend to drifting together until the conductive Ti phase between them is not oxidized, once the metal phase is oxidized, the thickness of the oxide layer increases and the strength of the electric field in barrier layer drastically decreases, which resulted in the decrease in the migration rate of O2− ions through the film. On the other hand, the diffusion lengths of O2− are nearly equal for all pores and the structure tends to form a spatially ordered array with maximal packing density . In summary, the mechanism of TNT formation consists of the following steps (shown in Fig. 10): (a) formation of oxide layer, (b) pore formation and deepening of the pore, (c) incorporation of adjacent small pores into a big pore, (d) earlier nanotube arrays formation, and (e) formation of perfect nanotube arrays .
4 Properties OF TNT
4.1 Phase Structure
The structure of the as-grown TNT is amorphous or crystalline, and this is strongly dependent on the specific electrochemical parameters such as applied potential, anodization time, and/or the sweep rate of the potential ramp. For example, the structure of the oxide films on Ti has typically been reported to be amorphous at low voltages; crystallization takes place at higher voltages . Depending on the anodizing conditions, the crystal structure has been reported to be anatase , mixture of anatase and rutile , or rutile .
Mostly, the as-formed TNT has an amorphous structure [183, 184, 185]. Several studies have shown that the amorphous phase of tubes can be converted into anatase phase at higher temperatures (at 280 °C) in air  or a mixture of anatase and rutile [61, 186] at temperatures higher than 450 °C. It has also been reported that under certain conditions, nanocrystallites were present in the as-formed tubes .
The TNT obtained directly after anodization is amorphous and not photoactive. Therefore, it is necessary to transform the amorphous TNT into crystalline form of TNT for the use in a wide range of applications. High-temperature annealing has been regarded as an effective route to induce crystalline formation of the as-prepared TNT, converting them into anatase or rutile phase . To convert the amorphous tube into crystalline anatase or rutile phase, usually annealing is necessary, because annealing operations at several hundred degree Celsius are necessary after the anodization process. A large amount of energy is consumed in the formation of the crystalline TNT on lower thermostability material substrates .
Nanotubular integrity is maintained at annealing temperature lesser than 600 °C, whereas annealing in air for 3 h at a higher temperature (greater than ≥600 °C) resulted in collapse of nanotube bundles. The rutile phase emerges near 480 °C. Above this temperature, the anatase phase gradually transforms into rutile phase. At 700 °C, the amorphous Ti becomes suppressed and transforms to crystalline Ti [189, 190]. Moreover, annealing at 700 °C favors sintering and tends to destroy nanostructures completely. It is also reported elsewhere that nanotube structure was found to be stable up to approximately 550 °C. Above this temperature, nanotubes start collapsing and are destroyed . Jaroenworaluck et al.  also reported similar observation at 600 °C. Hence, annealing temperature influences on the resistance of the nanotube structure, the annealing temperature increases the resistance of the nanotube decreases. The anatase tubes show considerably lower resistance than rutile containing tubes. Long-time annealing shows significant decrease in resistance . After calcinations at 400 °C, TNT powder (prepared through rapid break down anodization) lost their tubular morphology and became spherical nanoparticles with the crystallites size of approximately 10–20 nm .
Some studies reported that the as-anodized TNT species exist slight anatase phase, which may due to the following reasons: (1) the outward migration of Ti4+ from the substrate and inward migration of O2−/OH− to form crystalline oxide in a ratio that favors crystal growth, (2) the internal stress relaxation of the oxide and the tube wall became thinner by dissolution after long-time anodization, and (3) faster ion mobility in the more water containing electrolyte can offer adequate ions that favor crystal growth . Higher voltages can promote the crystal phase transformation spontaneously without the application of heat treatment .
Annealing not only influences the phase change, but it as well affects the morphology of TNT. It is reported by Lim et al  that there were no changes in the morphology of TNT up to the annealing temperature of 500 °C. At the annealing temperature of 600 °C, a slight decrease in the diameter and length and an increase in the wall thickness were observed. The wall thickness of TNT increases after the annealing process, which is due to the growth of crystallite on the walls. Generally, the nanostructured TNT with high surface area is thermodynamically metastable, which is prone to solid-state sintering at higher annealing temperatures. This subsequently led to grain growth, densification, and increase in the tube wall thickness . Since sintering is an activated process involving mass transfer process, a higher annealing temperature and/or prolonged annealing process favor more sintering, which led to complete destruction of TNT structures . In other words, the change in the morphology of TNT with annealing temperature could be associated with the excessive Ti ion diffusion along the tube walls, which induces the oxidation, and thus thickens the tube wall thickness [167, 199].
4.2 Wettability of TNT
The water contact angle of a biomaterial is a complex issue and can be affected by many factors, such as the manner of surface preparation, surface roughness, surface compositions, and chemical states [41, 42].
Brammer et al.  developed TNT with different diameters such as 30, 50, 70, and 100 nm. They found that the contact angle reduces by increasing the tube diameter, i.e., the contact angle was found to be 11, 9, 7, and 4 for 30, 50, 70, and 100 nm diameter tubes, respectively. This is due to the fact that higher the diameter of the tubes, higher the absorbance of the liquid. Similarly, Narayanan et al. reported that the water wetting angles (hydrophilic nature) increased with pore diameters which will have an important consequence on the adhesion of cells to the nanotube containing surface. Cells tend to attach better to hydrophilic surfaces than to hydrophobic surfaces .
It is reported elsewhere that the anodization voltage was found to have a strong effect on the resulting contact angle; at the applied voltage of 25 V, the contact angle was found to be 41.4°, whereas at the voltage of 40 V, the contact angle was increased to 13.8°. Also,, the contact angle for the freshly prepared electrolyte was found to be 51.9°, while the used electrolyte led to the highly increased contact angle with the value of up to 95.0° ± 14.8°, which clearly indicating the very low wettability of the film . The nano/micro interface displayed a considerable hydrophilic property; the water contact angle (CA) was only 23.3° ± 7.9°. Thus, this hydrophilic surface is better for blood adsorption than a hydrophobic surface. It is well established from the literature that the as-formed TiO2 surface is often partially hydroxylated and is therefore of a polar nature, which leads to relatively small advancing contact angles. In addition, the TiO2 parameter would become dominant for very hydrophilic surface, which should contain a high number of Lewis acidic Ti–OH groups .
In addition, crystalline phase of TNT also plays a vital role in the surface wettability. Recently, Munirathinam et al.  reported that similar to the amorphous TNT, the annealed TNT also exhibited hydrophilic nature, which is mainly due to the presence of mixed crystalline phase (anatase and rutile). The hydrophilic behavior of amorphous TNT could be attributed to the density of hydroxyl group on their surface and the high polarity of O–Ti–O bond. Up to the annealing temperature of 450 °C, the TNT surface is super hydrophilic, when the annealing temperature was further increased, the hydrophilic nature started to decrease. This is because of the lack of hydroxyl group on the surface of TNT after annealing at higher temperatures (above 450 °C). The surface hydroxyl can easily combine with water molecule, thus, formed hydrogen bond resulting in good wettability .
4.3 Surface Roughness
Generally, biologically active implant materials have enhanced surface roughness, which was one of the important factors in providing better cell response to implanted materials . Lu et al.  studied the effect of the surface roughness on the TNT formation during the anodization, which showed that the nanotubes formed from the as-received and chemically polished samples have rough surfaces, as a result, the corresponding nanotube morphology and bottom oxide barrier layer are non-uniform.
The average surface roughness of TNT prepared using ethylene glycol and ammonium fluoride electrolyte was found to be 360 nm . Anodization increased the surface roughness by forming a porous layer on Ti surface. It has been reported elsewhere that the surface roughness of TNT with the diameter of 30, 50, 70, and 100 nm was 13, 12.7, 13.5, and 13.2 nm, respectively . This result confirmed that the tube diameter has not played any role in altering the surface roughness of the TNT. It is reported elsewhere that the TNT showed nanorough (12.62 nm) surface and the anodization process did not create microtopographical differences, whereas it did create nanotopographical differences .
The inter-tubular spacing in the TNT has significant influence on the surface roughness . Annealing not only influences the phase change, but it as well affects the lateral spacing between the TNT which is attributed to the crystallization and crystal growth . It is reported by Lim et al.  that there were no changes in the morphology of TNT up to the annealing temperature of 500 °C. A slight increase in the wall thickness (laterally attached together), in other words, a decrease in the lateral spacing was observed at the annealing temperature of 600 °C. The lateral spacing of TNT decreases after the annealing process, which is due to the growth of crystallite on the walls. Generally, the nanostructured TNT with high surface area is thermodynamically metastable, which is prone to solid-state sintering at higher annealing temperatures. This subsequently led to grain growth, densification, and decrease in the lateral spacing . In addition, annealing at higher temperature leads to the formation of barrier layer between the tubes and the underlying Ti substrate . In other words, decrease in the lateral spacing of TNT with annealing temperature could be associated with the excessive Ti ion diffusion along the tube walls, which induces the oxidation, and thus thickens the tube wall thickness, i.e., decreases the lateral spacing . Besides, the distance between the WE and CE also influences the inter-tubular spacing, which was already discussed in the previous section (Sect. 2.7).
5 Biomedical Applications
According to many researchers, the TNT might be used in drug-eluting stents and for the local release of antibiotics , drugs, or growth factors from orthopedic and dental implants . Hence, this section provides the detailed information of TNT in the biomedical field.
5.1 As Orthopedic and Dental Implants
A common treatment for bone fracture is the implantation of a mostly metallic orthopedic prosthetics. These prosthetics help in healing bone non-unions and allow patients to partially regain their function. However, today’s orthopedic implant materials do not allow patients to return to their normal, daily active lifestyles that they had before fracture. Specifically, it has been reported that the average lifetime of orthopedic implants is only 10–15 years. This means that those who are young and receive a traditional orthopedic implant will have to undergo several more painful and expensive revision surgeries to replace such a failed orthopedic implant. Although controversial several factors such as incomplete, prolonged osseo-integration (i.e., lack of bonding of an orthopedic implant to juxtaposed bone) and severe stress shielding (due to differences in mechanical properties between an implant and the surrounding bone) lead to implant failures . Therefore, a great amount of efforts from biomedical researchers worldwide have given on improving the design and manufacture of orthopedic implants to last longer in the body. While many have attempted to alter orthopedic implant chemistry (from metals to ceramics to polymers), recent discoveries have highlighted that nanotechnology may universally improve all materials used for re-growing bone. The use of nanostructured materials has been proposed to solve some of the problems currently associated with orthopedic implants .
As a biocompatible material, Ti and its alloys are extensively used in orthopedic and dental implants mainly because of its optimal mechanical properties in load-bearing applications . However, insufficient new bone formation is frequently observed on Ti which sometimes leads to implant loosening and failure. For this reason, TNT with good bioactivity has been developed by anodic oxidation which may be a simple method to modify the surface of Ti implants to enhance bone-forming function, thereby increasing orthopedic implant efficiency . There have been several studies [216, 217] reported on TNT for bone tissue engineering applications.
The presence of HAp coating promotes osteo-conduction. Surface treatments such as roughing by sand blasting, formation of anatase phase TiO2, HAp, and Ca10PO4·2H2O coating have been utilized to further improve the bioactivity of Ti and enhance bone growth. Heat-treated TNT, when soaked in simulated body fluid (SBF) solution is completely covered with HAp layer, and thereby provides excellent bioactivity . It is well established that the anatase phase TiO2 is much more efficient in nucleation and growth of HAp than the rutile phase TiO2 presumably because of the better lattice match with HAp phase. The formation of bone growth-related material such as the calcium-phosphate HAp is an important issue for orthopedic and dental implants. Bone is a calcium-phosphate-based mineral which contains ~70 % HAp-like material with remainder consisting mostly of collagen .
Treating TNT with NaOH solution induces the growth of nanosized HAp when subjected to SBF. Furthermore, nanophase ceramics such as HAp, Al2O3, and TiO2 enhance long-term osteoblast functions relative to typical microstructure forms. Thus, the HAp forming and bone-bonding ability of Ti bone implant materials may be significantly enhanced by anodically oxidized nanotubes on the surface of Ti . The TNT is adherent well to Ti implant surface and can be useful for accelerated bone growth in orthopedic and dental applications. Advantage of strong mechanical adherence, the gaps present between adjacent TNT, may also be useful as a pathway for continuous supply of the body fluid with ions, nutrients, and proteins. This is likely to continue positively to the health of the growing cell [220, 221]. The TNT with Ca was also developed, where it was used for bone repair in filling defective areas of bones. Newly formed bone was found around TNT with Ca surface after being implanted in the femur of a rat for 7 days. This phenomenon indicated that Ca-TNTs induced excellent bone tissue regeneration at implantation . Very low adhesion strengths were reported for electrochemically produced HAp coatings. Plasma-sprayed HAp coating has also been reported to have tensile adhesion strengths lower that 10 MPa, and their heterogeneous structure was reported to cause poor adhesion. Poor adhesion of TNT is attributed to the stirring condition of the electrolyte .
5.2 As a Corrosion-Resistant Materials
5.3 As Blood Clotting and Anti-microbial Agent
The blood and tissue compatible coatings on surgical tools as well as artificial bone and dental implants are of paramount importance. The TiO2 helps to retain the natural forms of proteins and also minimize unwanted platelet activation due to its passive surface and chemical stability. The TNT is used as an adhesion and growth support platform for bone and stem cells [229, 230]. Gauze bandage surfaces decorated with TNT could be used to improve the rate of blood clot formation and the strength of the resulting clot .
Silver (Ag)-doped TNT has good biological applications. However, Ag is probably the most powerful anti-microbial that exhibits strong cytotoxicity toward a broad range of micro-organisms and remarkable low human toxicity compared to other heavy metal ions . Recently, studies on cell interaction with TNT show cell adhesion, proliferation, and migration, and are significantly affected by the nanotube size . The TNT can improve osteoblast attachment, function, and proliferation. Furthermore, these surfaces also exhibit very low immunogenicity, eliciting low levels of monocyte activation, and cytokine secretion . Others [233, 234] suggest that nanotubes are also used in vascular applications, enhancing endothelial cell ECM production and mobility. Recently, Lan et al.  decorated the TNT with Ag by e-gun vacuum evaporation system and studied the influence of tube diameter on human nasal epithelial cell. The results showed that the Ag-coated TNT with the diameter of 25 nm revealed antibacterial effect against P. aeruginosa.
5.4 In Cancer and Tumor Therapy
Most recently, researchers found that the TNT is a potential drug delivery vehicle in nanomedicine, which is used as a nanovehicle for cancer drug delivery. Generally, nanotubes have significant advantages over nanoparticles in a medicine carrier. In contrast to the spherical nanoparticles, the geometry of nanotubes must allow them to be multiply functionalized by different molecules that allow the nanovehicle to carry the drug more efficiently to target for the purpose of cancer therapy. The exploration of nuclear DNA-targeting nanotubes carrying anti-tumor drugs is of particular importance. Undoubtedly, it is one of the difficult challenges in the emerging area of cancer nanotechnology. The TNT is able to cross karyotheca and enter the nucleus, hence, it is a promising future nanovehicle for DNA-targeting drugs in cancer therapy [236, 237].
The self-organized, highly ordered tubular architecture of anodic TNT has been shown to be an excellent substrate for adherence, spreading, and growth of living cells, which promote cellular activity. When the tube morphological characteristics are approximately tuned, most importantly, the anodic TNT can act as efficient photocatalysts for the photo-induced killing of cancer cells . The dimensions of the nanotubes play a vital role for cell adhesion and spreading. A possible application of the nanotubes may be anticancer treatment, where isolated tubes are administrated to a tumor, followed by a focused UV-light exposition of the tumor . Chen et al.  incorporated selinium and chitosan onto TNT for the anticancer treatment. The results indicated that TNT with selenium and chitosan substrates promoted the biological functions of healthy osteoblasts and inhibited the growth of cancerous osteoblasts. More importantly, in vitro antibacterial assay revealed that the TNT with selenium and chitosan substrates had good antibacterial property.
5.5 In Drug Delivery
In recent years, controlled drug delivery applications have gained increasing attention and the rapid expansion in the advanced materials, and technology has resulted in a remarkable progress in their development. The TiO2 is a good candidate for drug-eluting implant coating as it is highly biocompatible and able to control protein and antibiotic . Benefiting from large surface area, excellent biocompatibility, and one-end open feature, the TNT has been applied in drug delivery [241, 242]. A large number of implants in dentistry and orthopedics are made from Ti alloys, and the possibility of using TNT to deliver drugs and other compounds from these implants appear to be quite attractive. Local delivery of drug from an implant surface may be desirable as it can reduce the amount of drug requirement and may also reduce the systemic side effects.
Moseke et al.  reported that the TNT can be used as a long-term drug delivery system for anti-microbial agents. It has been reported elsewhere that the chemical functionalized groups may act as an inbuilt regulator for achieving different drug dosages. Modulation of surface chemistry and kinetics of TNT alters both the morphological parameters (e.g., surface area and pore dimensions) and the drug loading percentages. The loading amount gets reduced with hydrophobic surface, whereas it increases with hydrophilic moieties. This in turn influences the release rates, thus, varying from fast release with hydrophobic functionalization to very slow release with hydrophilic functionalization. These chemical functionalized groups may act as an inbuilt regulator for achieving different drug dosages. The main driving force for the drug encapsulation arises from hydrogen bond interaction between the drug molecule and the –OH groups present in the pore walls of TNT. Given that the drug and pore wall interaction are high, the release rate is low and vice versa. If the surface of TNT is modified with the hydrophobic groups like –CH3 and impregnated with drugs like ibuprofen, the interaction between the drug and the tube walls gets decreased leading to low loading percentage, which in turn increases the release rate of drug .
It has been reported that the nanotube height and the loading solution concentration have more influence on drug release, whereas the crystalline structure of the nanotubes does not influence the amount of drug released . It has also been reported that the TNT could maintain the bioactivity of the loaded drug and dictate the biological responses of mesenchymal stem cells . Wang et al.  developed TNT with P25 nanoparticles for the effective delivery of ibuprofen drug. They found that the TNT with P25 nanoparticles could improve the drug adsorption capacity and sustained release of drugs. The TNT with biodegradable polymers like chitosan and PLGA has extended drug release property, biocompatibility for human osteoblasts, and potentially improved antibacterial properties. This kind of polymer-modified TNT is capable of delivering the drug to a bone site over an extended period with predictable kinetics .
In this article, we reviewed the synthesis, formation mechanism, various parameters, which affects the tube formation mechanism and properties of TNT and their wide range of applications in the biomedical field. The tube length, thickness, and diameter were varied according to the pH, electrolytes, anodization potential, and time. The as-prepared nanotubes have amorphous structure and can be transformed into crystalline by annealing at higher temperature. The crystalline nanotubes were often used for the bio-implant application purposes. Today, nanotubes are used as orthopedic and dental implants, blood clotting, and anti-microbial agents and also used as an effective drug delivery vehicle for the cancer therapy. By using the TNT, the field of nanomedicine has a bright future with the emergence of several promising approaches for drug delivery and cancer treatments.
Compliance with Ethical Standards
Conflict of Interest
There is no conflict of interest in this article.
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