Article
In-vitro biocompatibility and corrosion resistance of strontium incorporated TiO2 nanotube arrays for orthopaedic applications
Journal of Biomaterials Applications 2014, Vol. 29(1) 113–129 ! The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328213516821 jba.sagepub.com
K Indira1, U Kamachi Mudali2 and N Rajendran1
Abstract This article investigates the in-vitro biocompatibility and corrosion behaviour of strontium ion incorporated TiO2 nanotube arrays formed by anodization method for orthopaedic applications. The morphological studies were carried out using field emission scanning electron microscopy, atomic force microscopy, attenuated total reflectance fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and thin film X-ray diffraction techniques. The morphological investigation indicated that the length and the average diameter of nanotube were 2.1 0.3 mm and 110 4 nm, respectively. The wettability measurements showed that the TiO2 nanotube arrays have super wettability, as well as, strontium ion incorporated TiO2 nanotube arrays exhibited hydrophilic behaviour. Excellent in-vitro bioactivity was observed for TiO2 nanotube arrays with strontium ions. Electrochemical studies in Hank’s solution showed that the TiO2 nanotube arrays with strontium ions have enhanced corrosion resistance. Keywords Anodization, TiO2 nanotubes, dip coating, Hank’s solution, biocompatibility
Introduction Titanium (Ti) and its alloys are widely used as biomedical implant materials owing to their high corrosion resistance, excellent biocompatibility, low specific gravity and long fatigue life.1–6 Basically, metal oxides with nanostructures are capable of improving the longevity of implants, as nanoscale surfaces exhibit uniform surface energy by the interaction with bone cell. For instance, one-dimensional TiO2 nanostructures such as nanotubes and nanowires have appreciable properties such as specific surface area and mechanical strength.7 Thus, TiO2 nanotube arrays (TNTA) have gained considerable attention during the last 15 years.8 Furthermore, nanostructures can be used as a template for the deposition of other materials into the pores, followed by selective etching of the TiO2 mold.9 The TNTA developed posses higher adhesion strength. The TNTA produced in anodization is following selforganization behaviour, thus the tubes are directly attached to the titanium surface and are already electrically connected to the substrate.10 The tubes are growing directly from the titanium substrate, it is considered to be a part of the substrate. Hence, the
adhesion strength of the TNTA is good, which is also reported in the earlier reports.11 In recent times, incorporation of biocompatible elements such as Sr12 and Ag13 into TNTA has triggered enormous attention in biomedical field. Among all, strontium (Sr) has considerable application in biomedical field due to controlled drug delivery system. Oral administration of drug can increase the drug concentration in the blood plasma. On the other hand, a controlled drug delivery system on the biomedical implants can offer a sustained complement of the element or drug. Hence, Sr can be capable of delivering the drug in controlled manner than other elements. Therefore, the release of Sr from the nanostructured surface of Ti implant not only encourages nanosize 1
Department of Chemistry, Anna University, Chennai, India Corrosion Science and Technology Group, Indira Gandhi Center for Atomic Research, Kalpakkam, India 2
Corresponding author: N Rajendran, Department of Chemistry, Anna University, Chennai 600 025, India. Email: [email protected]
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effects but also facilitates in-situ Sr delivery to accelerate osseointegration and favourable osteogenic effects.12 The Sr loaded into TNTA is released to the implant tissue interface locally, which directly enables effective absorption by tissues in the vicinity, thereby promoting bone formation and suppressing bone resorption.14 Hence, the aim of this study is to incorporate Sr ions into TNTA and to investigate the in-vitro and electrochemical behaviour of Sr ion incorporated TNTA (Sr-TNTA) in Hank’s solution. Incorporation of Sr ion was done by simple dip-coating method. The surface morphological, phase structural and topographical properties were observed using microscopic techniques such as field emission scanning electron microscopy (FESEM), thin film X-ray diffraction (TF-XRD) and atomic force microscopy (AFM), respectively. Electrochemical characterization studies were carried out in Hank’s solution using electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization measurements.
Materials and methods Preparation of TNTA by anodization Prior to anodization, the Ti sheets (99.9%, M/s. Ti Anode Fabricators Pvt. Ltd., Chennai, India) were grounded on both sides with silicon carbide paper up to 1000 grit. Upon grinding, the samples were ultrasonically cleaned in a mixture of acetone and 2-propanol for 10 min, then pickled in a mixture of 0.9 M HF and 3.0 M HNO3 for 1 min and dried in air at room temperature. A two-electrode cell set up consisting of platinum cathode of size 1.08 cm3, Ti anode of size 0.11 cm3 were employed for anodization. The anodization was carried out with slight modification in the earlier report.15 The electrolyte used was a mixture of 0.26 M NH4F and 14.5 M ethylene glycol. The constant potential of 50 V was applied for 2.5 h at room temperature with magnetic agitation using DC power supply (M/s Aplab, Model H0615, India). The TNTA was then annealed at 500 C for 2 h. Sr ions were incorporated into TNTA by immersing the anodized specimens in 0.02 M Sr(NO3)2 solution for 10 min and this process was repeated three times. The dip coater used for the present study was Single dip coater (Model SDC 2007C, M/s Apex Instruments & Co., India). The dip-coated specimens were annealed at 500 C for 2 h.
In-vitro studies The in-vitro bioactivity of the specimen was studied by immersing the anodized specimens in Hank’s solution for 7 days and examining the apatite (HAp) forming
ability of the Sr-TNTA. Hank’s solution was prepared as described in the earlier report.3 After immersion in Hank’s solution for 7 days, the specimens were rinsed with distilled water and dried at room temperature. The wettability of the specimens were determined using water droplet contact angle measurement (OCA 15EC, data physics instruments, Germany) with a dosing volume and dosing rate of 10 mL and 1 mL/Sec, respectively.
Surface characterization The surface morphology and elemental composition were characterized by FE-SEM (GEMINI-Supra 55, Germany) equipped with energy dispersive X-ray spectroscopy (EDS). In order to find out the functional groups present on the surface, Attenuated Total Reflectance Fourier Transform Infrared (ATR-IR) spectroscopy was carried out in the frequency range from 400 cm–1 to 4000 cm–1 (Perkin Elmer spectrum 2). The X-ray Photoelectron Spectroscopy (XPS, SPECS made XPS with Al K-a PSOI BOS-150, Germany) was done using Al Ka radiation (1486.71 eV) as the excitation source. The binding energies of target elements (Ti 2 p, O 1 s, Sr 3 d, Ca 2 p and P 2 p) were obtained at the pass energy of 12 eV with the resolution of 0.1 eV. TF-XRD measurements were performed with Bruker D8 Discover (TF-XRD, Germany) using Cu Ka radiation. The surface topography of the samples was seen using AFM (NT-MDT SPM, Russia) in contact mode for a scan area of 20 mm 20 mm and the surface roughness was measured from the AFM topographs using an image analysis tool called ‘NOVA Image Analysis software 1. 0. 26. 1443,’ which is associated with the AFM.
Electrochemical characterization Electrochemical experiments were performed using a conventional three-electrode cell assembly maintained at 37 C. The electrodes namely reference, counter and working electrodes used in the experiments were saturated Ag/AgCl, Pt sheet and test specimens, respectively. Potentiodynamic polarization measurements were carried out for untreated Ti, TNTA and Sr-TNTA after immediate and 7 days of immersion in Hank’s solution using Solartron 1287 Electrochemical Interface. The electrode potential was anodically scanned at a scan rate of 10 mV min–1. All the measurements were carried out in aerated and non-stirred conditions. The corrosion current density (icorr) and corrosion potential (Ecorr) of each sample was calculated from polarization curves with the support of Cview software.
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EIS measurements were carried out using Solartron 1255 frequency response analyzer (FRA) with Solartron 1287 electrochemical interface associated with the standard three-electrode cell assembly as mentioned earlier. All the experiments were carried out in the frequency ranging of 104 to 10–2 Hz, by superimposing an AC voltage of 10 mV amplitude with the data density of 5 points per decade in Hank’s solution. The impedance response obtained over the applied frequency range was represented by Bode plots. For the analysis of impedance results, equivalent circuit was used.16,17
electronic charge transfer, but the gas bubbles were minimum at the end. Figure 2 shows the current transient (I-t) curves recorded during anodization. The initial current was 0.022 mA, which was due to the surface oxidation conditions. The initial higher current drops with time (0–3 s) (inset in Figure 2) indicates the formation of oxide/hydroxide layer over the Ti surface and the subsequent current rise (3–36 s) is due to pitting/ dissolution of the oxide layer by fluoride ions.18 Subsequently, in the next stage, the observed current drop (36–1000 s) indicates the overlap of the above two processes i.e. oxide layer formation and dissolution. At last, the current reaches a steady state which is attributed to the stable film on the surface. It has been reported that, higher current is due to the fast field aided F– transport through the growing oxide layer, which promotes the dissolution of oxide layer.19 Current density is directly related to the tube length, thus, higher current density leads to longer tubes.9
Results and discussion Outlook of the present investigation The schematic representation of HAp growth over Sr-TNTA takes place in three steps and is shown in Figure 1. Initially, anodization of Ti in a mixture of NH4F and ethylene glycol develops the TNTA (first step), in the second step Sr-TNTA layer was formed over the nanotube arrays by dipping in strontium nitrate solution followed by annealing. In the third step, HAp deposits over Sr-TNTA, which has been proved with the support of different studies in the following sections.
Formation mechanism of TNTA During the formation of TNTA, ammonium fluoride and water are ionized into NH4þ, F–, Hþ and OH–, respectively. Ethylene glycol does not participate in the reaction; instead it acts only as a solvent. The possible reaction behind the formation of TNTA is as follows:
Current-time behaviour during anodization
Ti þ 4H2 O
Visible changes were observed at the anode as well as at the cathode during anodization. Different colours form on Ti surface, i.e. from grey to pale green to blue, which indicate the change in the thickness of the oxide layer formed on the surface. Hydrogen gas bubbles evolved throughout anodization at the cathode, which was very intense at the beginning indicating that there was
NH4 F 4 TiðOHÞ4 þ2H2 " HOðCH2 Þ2 OH
Ti þ 4H2 O þ 4NH4 F
" HOðCH2 Þ2 OH 4 TiðOHÞ4 þ4NHþ 4 þ 2F2 " þ2H2
ð2Þ
The F2 and H2 gas evolved at the anode and cathode, respectively. Finally, the product obtained was Ti(OH)4
First step
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TiO2 nanotube arrays (TNTA)
TI substrate
ð1Þ
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Oxide layer Immersion in Hank’s Isolution 7 days TNTA
Sr-TNTA
Sr-TNTA - HAp
Third step
Hydroxyapatite (HA) layer
Figure 1. Schematic illustration of HAp deposited TNTA after the incorporation of Sr ion. TNTA: TiO2 nanotube array.
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and not TiO2. This is in line with the findings of Shin et al.20 Therefore, the following steps were involved in the formation of nanotube arrays: (1) growth of oxide layer on the surface of Ti due to the interaction of O2– and Ti4þ ,21 (2) dissolution of oxide layer, (3) pit formation on the surface and (4) development of pores from the pits.22 In the presence of fluoride ions, the oxide layer partially dissolved along with continuous pit formation and finally, the pits were transformed into pores and simultaneously into tubes. Formation mechanism of Sr-TNTA involves the following reaction (equation (3)); which depicts the precipitation of SrTiO3. The divalent Sr reacts with the titanium hydroxide (Ti(OH)4) and precipitates out SrTiO3. Sr2þ þ TiðOHÞ4 ! SrTiO3 þ H2 O þ H2 "
ð3Þ
Surface characterization The FE-SEM images and EDS results of the specimen are shown in Figure 3(a)–(h). The surface morphology of untreated Ti reveals only the polishing grooves as shown in Figure 3(a). After anodization, nanotubes arrays (Figure 3(c)) with the average diameter and wall thickness of 110 4 nm and 15 2 nm,
respectively, were observed. It is clear from Figure 3(e) that tube structures were formed with a length of approx. 2.1 0.3 mm. From Figure 3(f), it is apparent that the tube mouth was open at the top and closed at the bottom. The average inter-tube diameter (distance between the centre of the adjacent tubes) was found to be 128 2 nm. After dipping the TNTA specimens into strontium nitrate solution, Sr ions cover the walls of the tube. In addition, some Sr ions were scattered like clusters over the top surface of the TNTA. The TNTA (Figure 3(g)) retained the original nanotube structure after the incorporation of Sr ions, however, minor decrease (20 5 nm) in the diameter (during the transformation of TNTA to Sr-TNTA, the diameters of the tubes were changed from 110 to 90 nm respectively) was observed, which was probably due to volume expansion during the transformation from titanium oxide to strontium titanate.14 It has been reported that the inter-pore distance, pore diameter and wall thickness depend linearly on anodization conditions.23 The EDS (Figure 3(b), (d) and (h)) results showed the presence of Ti, O and traces of F on TNTA surface. In addition to Ti and O, Sr is also present on the surface of Sr-TNTA, which confirmed the presence of Sr ions over the TNTA surface. It is reported in the literature that the mechanical properties of the nanostructure Ti is higher than the commercially pure Ti.24 Brammer et al.24 proved that the Ti surfaces with nanotubes interacted most
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Figure 3. FE-SEM images of (a) untreated Ti, (c) TNTA (e) cross sectional view of TNTA and (f) bottom view of TNTA and (g) Sr-TNTA and EDS spectrum of (b) untreated Ti, (d) TNTA and (h) Sr-TNTA.
efficiently with primary bovine chondrocytes by preserving the characteristic spherical morphology and increasing extracellular matrix bril secretion and biochemical production when compared to a plane Ti surface. Park et al.25 reported that the cell adhesion, spreading, and growth on surfaces of this nanometric scale was enhanced in comparison to plain TiO2 surfaces. It is reported elsewhere that the nanoscaled topography can guide enhanced cellular migration on the surface, promote differentiation and matrix production of bone cells and enhance short- and long-term
osseointegration both in vitro and in vivo.14 In the case of nanostructured TNTA, the Sr ions sat on the walls of the tube and bonded to the surface very strongly (since, TNTA has higher adhesion strength than bear Ti), whereas, in the case of plane Ti, the Sr ions were deposited on the compact oxide layer, where the adhesion strength is very poor. Hence, the nanostructured topography could reveal the favorable effect on Sr loading than the plane Ti. Figure 4(a)(d) presents ATR-IR spectra of the samples. The untreated Ti (Figure 4(a)) exhibited a
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weak band at around 440 cm–1, which can ascribe to Ti-O bond originating from the native oxide layer. TNTA (Figure 4(b)) showed a triplet peak from 3448 cm–1 to 2864 cm–1, which are ascribed to v3 and v1 stretching modes of O-H bond (may be due to Ti-OH group).26 The peak at 920 cm–1 is assigned to O-O bond. After annealing (Figure 4(c)), the intensity of O-H bond gets reduced and the Ti-O bond becomes stronger. The Sr-O (Figure 4(d)) bond causes the vibration at 1360 cm–1,27 and the peak at 452 cm–1 is attributed to Ti-O bond. The intensity of the O-H bond diminishes in Sr-TNTA which may due to the annealing effect. ATR-IR confirmed the presence of Sr-O
bond. In addition, the presence of Sr over TNTA surface is further confirmed using XPS and TF-XRD. Chemical state of the elements present on the surface was studied using XPS and is shown in Figure 5(a)–(e). The Ti 2 p signals in TNTA (Figure 5(a)) exhibits two dominant peaks, which corresponds to Ti4þ/TiO2 (Ti 2p1/2 at 465.2 eV and Ti 2p3/2 at 459.4 eV).28 In the O 1 s spectrum (Figure 5(b)), the Ti-OH/Ti-OH2 on TiO2 surface were observed at 530.8 eV and 532.0 eV, respectively.29 The Ti 2p1/2 and Ti 2p3/2 spectrum in Sr-TNTA has the binding energy values of 464.6 eV and 458.6 eV (Figure 5(c)), respectively, which corresponds to SrTiO3.12 The binding energy corresponding to O 1 s
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Figure 5. The XPS spectra of (a, b) TNTA and (c–e) Sr-TNTA.
at 530.0 eV (Figure 5(d)) is attributed to SrTiO3 and 531.3 eV corresponds to Ti-OH group indicating the presence of both TiO2 and SrTiO3 on the surface. The binding energy values at 134.6 and 136.4 eV (Figure 5(e)) were assigned for the Sr 3 d from SrTiO3.30 The crystal structure of as anodized TNTA, annealed TNTA and Sr-TNTA were characterized by TF-XRD and are shown in Figure 6(a)–(c). The diffraction pattern of TNTA shows only the Ti peaks which confirms that the as-anodized TNTA was in amorphous phase.22 The annealed TNTA (Figure 6(b)) exhibited the characteristic diffraction peaks of anatase phase of TiO2 at 2y values of 25.4 and 48.1 (JCPDS No.: 21-1272). The Sr-TNTA were composed of SrTiO3 (JCPDS, card no: 05-0634) and anatase phase of TiO2 (JCPDS, card no: 21-1272), which is in accordance with the XPS results. Anatase peak emerge, implying that part of Ti-O barely reacted with Sr and formed SrTiO3. The rest of the amorphous oxide has not reacted and has been converted into crystalline oxide (anatase) after heat treatment.28
Surface roughness and wettability Several factors such as surface composition, roughness and wettability play a significant role in implant tissueinteraction and osseointegration. Figure 7 shows the bar chart representation of roughness results, which showed that the average surface roughness of untreated
Ti, TNTA and Sr-TNTA were found to be 152, 360 and 125 nm, respectively. The roughness measurements showed that TNTA possess enhanced surface roughness than other samples, which may be attributed to the formation of adherent nanotube layer on Ti surface. The Sr-TNTA has very low roughness compared to the TNTA, which is probably due to the incorporation of Sr ions into the TNTA surface. It has been reported elsewhere that increase in thickness of the anodic layer increases the roughness as well as corrosion resistance of the material.31 Lu et al.9 studied the effect of the surface roughness on the TiO2 nanotubes formation during the anodization, which showed that the nanotubes formed from as-received and chemically polished samples have rough surfaces. As a result, the corresponding nanotube morphologies and the bottom oxide barrier layer are non-uniform. Generally, for bio-active implant materials, enhanced surface roughness is one of the most important key factors to provide better cell response. It has been reported that, besides surface chemistry and crystallinity, surface roughness influences the cellular response, enhancing cell adhesion and proliferation.32 Lower surface roughness gives a lesser bone contact,33 and the higher surface roughness provides not only an early fixation but also better mechanical stability for implants, which would result in good mechanical interlocking between the implant surface and bone ingrowth.34 The surface roughness of 10 nm to 10 mm
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Figure 7. Bar diagram representing the results of surface roughness. TNTA: TiO2 nanotube array.
can induce the apatite growth, thus improving the bioactivity of the material.35,36 It is reported elsewhere that the surface architecture at nanoscale may be one of the major factors influencing bacterial cell attachment to the surfaces.37 It is conceivable that the film with high roughness would have more adsorption sites of hydroxyls on the TiO2 surface, thus, the hydroxyl groups
combines with the positively charged calcium ions, which in turn combines with phosphate ions and form Hap.38 Park et al.39 reported that Sr incorporation into titanium oxide layer further enhance the cell attachment, feasibility of hydrophilic and micro-rough Ti surface. In order to achieve favourable implant bone healing, micro-rough surface property is one of the desirable factors. Hence, from the roughness value of Sr-TNTA, it is clear that it has mirco-rough surface, which is considered potentially favourable for implant materials. Contact angle is indirectly related to surface energy. Lower the contact angle, higher will be the surface energy.40 Higher surface energy materials favour the formation of HAp in biological solution. Super wettability of the specimens, with high surface energy, can induce HAp formation. Figure 8 shows the pictorial representation of the contact angle measurements of the specimens. The contact angle for the untreated Ti was 86 . Super wettability was observed for TNTA whereas the contact angle for Sr-TNTA was found to be 26 , which may be due to the presence of partial TiO2 on their surface. The hydrophilic nature of the TNTA was attributed to the capillary effect of the nanotubes.41 Improved wettability i.e. low contact
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Figure 8. Pictorial representation of contact angle results of the specimens. TNTA: TiO2 nanotube array.
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In order to assess the biocompatibility, the specimens were immersed in Hank’s solution for 7 days. Figure 9(a)–(i) displays the FE-SEM morphology and EDS spectra of untreated Ti, TNTA and Sr-TNTA after immersion in Hank’s solution for 7 days. Cluster-like particles were observed to be non-uniformly deposited over the untreated Ti surface (Figure 9(a) and (b)) after 7 days of immersion in Hank’s solution. Globular particles covered the TNTA (Figure 9(d) and (e)) layer whereas in the Sr-TNTA surface trabecular bone like morphology was observed uniformly all over the
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surface (Figure 9(g) and (h)). The EDS results (Figure 9(c), (f) and (i)) confirmed the presence of Ca and P over the surface (main elements in HAp), which correlated well with the results of ATR-IR, XPS and TF-XRD. From the images it is obvious that the HAp deposition was observed not only in a small part of the SrTNTA surface but in the whole area of the sample. However, Sr-TNTA is less hydrophilic than TNTA which may due to the higher crystalline nature of the Sr-TNTA (observed from the TF-XRD pattern). Generally, the hydrophilic surface (contact angle less than 90) can provide better bioactivity.36 It is reported elsewhere that the Sr is a potent bioactive element in the enhancement of bone healing, hence, the incorporation of Sr incorporation promoted the attachment and proliferation of osteoblastic cells on calcium phosphate bone substitutes and HAp coatings in vitro.42
Capuccini and co-workers reported the substitution of Sr on HA coating towards the in vitro osteoblast and osteoclast response. They found that the presence of Sr in the coating enhances osteoblast activity and differentiation, while it inhibits osteoclast production and proliferation.43 Also, it is reported by Ni et al.,44 that Sr-containing HA bone cements reduced the possibility of wear debris at the bone–cement interface, stimulated new bone formation and better physiological stress transfer between the implant and host bone in vivo. Osteoblastic differentiation from human mesenchymal stem cell (hMSCs) is an important step of bone formation. In order to study the effect of Sr on hMSCs and osteoblastic lineage, Sila-Asna et al. has studied the in vitro induction of hMSCs by using strontium ranelate, a natural trace amount in water, food and human skeleton. They conclude from their studies that the Sr is an important factor for inducing mesenchymal stem
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The negatively charged sites present on the TiO2 surface attract the positively charged Ca2þ, thus, promoting the nucleation of HAp. In order to evaluate the oxidation state of the specimen after immersion in Hank’s solution for 7 days, XPS study was carried out for TNTA and Sr-TNTA, which is presented in Figure 11(a)–(f). The O 1 s spectrum of TNTA at 532.4 eV is deconvoluted into two peaks arising from C-O and/or hydroxyl group and 534.1 eV from phosphate group. The binding energy values of Ca 2 p (Figure 11(b)) are at 348.6 eV and 352.2 eV, which is attributed to calcium hydrogen phosphate. The P 2 p (Figure 11(c)) peak at 134.1 eV is from phosphate group. The O 1 s spectrum (Figure 11(d)) of Sr-TNTA is deconvoluted into two peaks with the binding energy of 530.7 eV and 533.3 eV, which corresponds to oxygen in phosphate (PO43–) and hydroxyl group, respectively.49 The Ca 2 p spectra of Sr-TNTA
348.6
Intensity, (a.u.)
Intensity, (a.u.)
O1s
SrTiO3 þ H2 O ! Sr2 þ TiO2 þ 2OH
348.2
P 2p
Ca 2p
351.9
132
134
136
Binding energy (eV) (f)
134.9
Intensity, (a.u.)
(a)
was also found into the HAp layer. A study by Coreno et al.48 showed that the formation mechanism of HAp layer over crystalline SrTiO3 is similar to that of crystalline CaTiO3. SrTiO3 acts partly as insoluble TiO2 and partly as soluble SrO. The equation (4) describes the subtle dissolution of SrTiO3:
Intensity, (a.u.)
cells to differentiate into osteoblasts with further enhancement on bone formation. In addition, they show strong evidence of bone structure stabilization by expressing genes related to bone formation at early day of cell differentiation.45 Thus, incorporation of Sr into TNTA is a successful approach to enhance the osteoconductivity of implant materials. ATR-IR spectra of untreated Ti, TNTA and SrTNTA on immersion in Hank’s solution for 7 days are shown in Figure 10(a)–(c). The peaks at around 799 and 735 cm–1 for untreated Ti are attributed to the presence of P2O74– (pyrophosphate) group.46 Characteristic peak of v3 PO43– group was observed at 1030 cm–1 for untreated Ti. In the TNTA and SrTNTA spectra, a broad absorption band around 3680 to 2690 cm–1 originates from the v3 and v1 stretching modes of O-H bond, and the band at 1650 cm–1 is ascribed to v2 bending mode of O-H bond. A small band at 1420 cm–1 arises from CO32– group and a strong band at 1032 cm–1 is attributed to v3 mode of PO43– group.47 The bands at 658, 596 and 554 cm–1 are attributed to the crystallized HAp layer. The peaks corresponding to the phosphates and carbonates are identified on the ATR-IR spectra, which confirmed the formation of HAp over the Sr-TNTA surface; which is further proved by XPS and TFXRD studies. However, trace amount of carbonate
344 346 348 350 352 354 356
Binding energy (eV)
Sr 3d/P 2p
133.7
130
132
134
136
Figure 11. The XPS spectra of (a–c) TNTA and (d–f) Sr-TNTA after immersion in Hank’s solution for 7 days.
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Binding energy (eV)
140
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Ti TiO2 SrTiO3 HAP
Intensity, (a.u.)
(b)
(c)
(d)
20
30
40
50
60
2 theta , (degree)
Figure 12. The TF-XRD patterns of TNTA and Sr-TNTA after immersion in Hank’s solution for 7 days: before heat treatment (a and b) and after heat treatment (c and d).
had the binding energy of 348.2 and 351.9 eV, which is ascribed to calcium phosphate.50 Sr 3 d and P 2 p (Figure 11(f)) are co-analyzed because of the overlap between the two (Sr 3 d and P 2 p). The binding energy value at 134.9 eV is attributed to Sr 3d3/2 and the binding energy value of 133.7 eV is assigned to phosphorous in PO43–.51 Hence, the XPS study confirms that Ca, P and Sr containing phases (Ca3PO4 and SrTiO3) exist on the nanotube surface. Figure 12(a)–(d) represents the TF-XRD pattern of TNTA and Sr-TNTA after immersion in Hank’s solution for 7 days. The characteristic 2y peaks of HAp observed at 28.4 , 31.7 , 32.9 , 42.1 , 46.7 and 49.1 correspond to (102), (211), (300), (311), (222) and (213) planes of TNTA. The TF-XRD spectrum of Sr-TNTA discloses the existence of HAp layer (JCPDS, card
no: 09-0432) on crystalline Sr-TNTA. After heat treatment (Figure 12(c) and (d)) the crystalline HAp peaks at 25.9 corresponding to (002) plane is clearly visible and all the peaks become sharper than the previous one. These results together with ATR-IR and XPS confirmed the growth of HAp over the surface.
Electrochemical characterization The potentiodynamic polarization curves of untreated Ti, TNTA, Sr-TNTA after immediate and 7 days of immersion in Hank’s solution are presented in Figure 13(a) and (b). The current density remained almost constant and extended over a wide range of potential for all the samples. Insignificant increase in current density after 2 V was observed for untreated Ti and TNTA,
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(a) 3
(b) 3
Untreated Ti TNTA Sr-TNTA
Potential, E/V vs Ag-AgCl
Potential, E/V vs Ag-AgCl
2
1
0
Untreated Ti TNTA Sr-TNTA
2
1
0
–1
10–7
10–6 10–5 Current density, i/Acm–2
10–4
10–8
10–7 10–6 Current density, i/Acm–2
10–5
Figure 13. Potentiodynamic polarization results of untreated Ti, TNTA and Sr-TNTA after immersion in Hank’s solution: (a) immediate immersion and (b) 7 days immersion. TNTA: TiO2 nanotube array.
Untreated Ti TNTA Sr-TNTA Simulated
–Phase angle, degree
80
60
40
(b)
Log / Z /,0hm cm2
(a)
104
103
102
20
101
0
(c)
103
104
Untreated Ti TNTA Sr-TNTA Simulated
80
60
40
20
10–2 10–1 100 101 Log f, Hz
102
(d)
103
104
Untreated Ti TNTA Sr-TNTA Simulated
105 Log / Z /,0hm cm2
10–2 10–1 100 101 102 Log f, Hz
–Phase angle, degree
Untreated Ti TNTA Sr-TNTA Simulated
105
104
103
102
0 10–2 10–1 100
101 102 Log f, Hz
103
104
10–2 10–1 100
101 102 Log f, Hz
103
104
Figure 14. The EIS results of untreated Ti, TNTA, Sr-TNTA specimens after immersion in Hank’s solution: (a and b) immediate immersion and (c and d) 7 days immersion. TNTA: TiO2 nanotube array.
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Rs
Rb Qb
(b)
Rs
Rp
Rb
Qp
Qb
(c) Rs
Ra
Rb
Qa
Qb
(d) Rs
Ra
Rp
Qa
Qp
Rb Qb
Figure 15. The equivalent circuit models of the specimens after immersion in Hank’s solution: (a) untreated Ti after immediate immersion, (b) TNTA and Sr-TNTA after immediate immersion and (c) untreated Ti after 7 days of immersion and (d) TNTA and SrTNTA after 7 days of immersion.
which may be due to electron transfer across the oxide layer.1 The current density was minimum (Figure 13(b)) for Sr-TNTA after 7 days of immersion in Hank’s solution, which is due to the formation of stable HAp layer over Sr-TNTA surface, which was already confirmed by FE-SEM-EDS, ATR-IR, XPS and TF-XRD studies. An obvious increase in current between 1 V and 2 V was observed for Sr-TNTA after immersion in Hank’s solution and also the current density is higher after 1.7 V compared to TNTA sample. This can be attributed to the structural changes of the passive layer or changes in the ionic or electrical conductivity of the layer.52 Figure 14(a)–(d) shows the EIS for untreated Ti, TNTA, Sr-TNTA after immediate and 7 days of immersion in Hank’s solution. The Bode-phase angle maximum for TNTA and Sr-TNTA at lower frequency region was –57 and –56 and higher frequency region was –58 and –34 , respectively. After 7 days of immersion, the phase angle plot for untreated Ti exhibited two distinct behaviours and attained a maximum value of –77 at the intermediate frequency region. The phase angle for the lowest frequency was –41 . Significant shift in the phase angle for Sr-TNTA specimens after immersion in Hank’s solution for 7 days showed three distinct phase angle behaviour at higher, intermediate and lower frequency regions compared to other specimens. The phase angle values were found to
be –10 and –65 at higher and lower frequency regions, respectively. The equivalent circuit models proposed to obtain the best fitted results are shown in Figure 15(a)–(d). After immediate immersion, untreated Ti showed single time constant whereas, TNTA and Sr-TNTA showed two times constants, which is attributed to the inner barrier layer and outer porous layer. Untreated Ti after 7 days of immersion in Hank’s solution showed two time constants, whereas the TNTA and Sr-TNTA after immersion in Hank’s solution for 7 days showed three time constants. It has been reported that nucleation and growth of bonelike apatite takes place on untreated Ti surface on prolonged immersion in SBF solution.52 The third time constant in the TNTA and Sr-TNTA after immersion may be due to the newly formed HAp layer, which has been confirmed by EDS, ATR-IR, XPS and TF-XRD characterization. The calculated impedance parameters are given in Table 1. It was observed that the Rb of the anodized samples (TNTA, Sr-TNTA and Sr-TNTA after immersion in Hank’s solution for 7 days) showed enhanced value than untreated Ti, which confirms that the anodization process increases the thickness of the barrier layer. The Rp value of Sr-TNTA was higher compared to TNTA, which indicated that the increased thickness of the outer layer is due to the incorporation of Sr ions
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Table 1. Electrochemical impedance spectroscopy (EIS) parameters of untreated Ti, TNTA and Sr-TNTA after immediate and 7 days of immersion in Hank’s solution. Specimen Immediate immersion Untreated Ti TNTA Sr-TNTA 7 days immersion Untreated Ti TNTA Sr-TNTA
Rs (k cm2)
Ra (k cm2)
Qa (Fcm–2Sn)
na
Rp (k cm2)
Qp (Fcm–2Sn)
np
Rb (k cm2)
Qb (Fcm–2Sn)
nb
0.01 0.06 0.02
– – –
– – –
– – –
– 3.64 6.25
– 8.95 10–7 4.10 10–7
– 0.81 0.88
225 1410 1350
4.20 10–5 2.40 10–6 1.24 10–6
0.92 0.82 0.89
0.04 0.01 0.03
0.02 9.10 15.60
1.71 10–7 1.39 10–7 1.45 10–7
0.83 0.80 0.86
– 5.28 6.94
– 6.10 10––7 3.16 10–7
– 0.81 0.89
550 1248 1235
4.16 10–6 1.76 10–6 1.44 10–6
0.87 0.85 0.83
Rs: solution resistance; Rb: polarization resistance of barrier layer; Qb: double layer capacitancee of barrier layer; Rp: polarization resistance of porous layer; Qp: double layer capacitance of porous layer; Ra: polarization resistance of HAp layer and Qa: double layer capacitance of HAp layer; TNTA: TiO2 nanotube array.
over the TNTA surface. The pathways for ion transportation were offered by rough and porous layer, which decreases their barrier functions.53 Significant increase of porous layer resistance after immersion in Hank’s solution for 7 days suggests excellent HAp growth, which in turn is related to better corrosion resistance of the material. High Rp values indicated high compactness and passivity of the surfaces. The Ra is higher for Sr-TNTA meaning that thick HAp layer was formed over the surface. Lower Qp value for Sr-TNTA after 7 days of immersion is due the wadding of nanotube surface by the newly grown HAp layer.54 The ‘n’ values of all the layers were close to 1 which means all the layers behaved as an ideal capacitor. Hence, Sr-TNTA is an interesting material which offers the required bio-compatibility in the body fluid, making it applicable for orthopaedic implants.
Conclusions In summary, Sr-TNTA was successfully fabricated on Ti substrate by dip-coating method of anodic TNTA. The nanotube morphology is retained with small reduction in the tube diameter after the complete incorporation of Sr ions over the TNTA tube walls. HAp grown Sr-TNTA is considered potentially encouraging material for implant application, which is evident from the roughness and contact angle results. In vitro experiments demonstrated that Sr-TNTA can accelerate HAp growth in Hank’s solution; electrochemical experiments indicated good corrosion resistance and biocompatibility of the Sr-TNTA. Hence, it can be concluded that Sr-TNTA is a viable alternative in orthopaedic applications to provide improved corrosion resistance and enhanced biocompatibility.
Acknowledgements The authors acknowledge the University Grants Commission - Department of Atomic Energy - Consortium for Scientific Research for funding this project. The authors also thank Dr. M. Kamruddin and Ms. Sunitha, Materials Science Group, Indira Gandhi Centre for Atomic Research, Kalpakkam for their assistance in FE-SEM and TF-XRD characterization respectively. DST-FIST and UGC-DRS are gratefully acknowledged.
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25. Park J, Bauer S, Mark VKD, et al. Nanosize and Vitality: TiO2 nanotube diameter directs cell fate. Nano Lett 2007; 7: 1686–1691. 26. You JH and Hsu KY. Influence of chelating agent and reaction time on the swelling process for preparation of porous TiO2 particles. J Eur Ceram Soc 2010; 30: 1307–1315. 27. Poth J, Haberkorn R and Beck HP. Combustionsynthesis of SrTiO3 Part II. Sintering behaviour and surface characterization. J Eur Ceram Soc 2000; 20: 715–723. 28. Zhang X, Huo K, Hu L, et al. Synthesis and photocatalytic activity of highly ordered TiO2 and SrTiO3/TiO2 nanotube arrays on Ti substrates. J Am Ceram Soc 2010; 93: 2771–2778. 29. Kuo YY, Li TH, Yao JN, et al. Hydrothermal crystallization and modification of surface hydroxyl groups of anodized TiO2 nanotube-arrays for more efficient photoenergy conversion. Electrochim Acta 2012; 78: 236–243. 30. Shkabko A, Aguirre MH, Marozau I, et al. Synthesis and transport properties of SrTiO3xNy/SrTiO3d layered structures produced by microwave-induced plasma nitridation. J Phys D: Appl Phys 2009; 42: 154–202. 31. Park IS, Woo TG, Jeon WY, et al. Surface characteristics of titanium anodized in the four different types of electrolyte. Electrochim Acta 2007; 53: 863–870. 32. Brammer KS, Oh S, Frandsen CJ, et al. Biomaterials and biotechnology schemes utilizing TiO2 nanotube arrays. In: Pignatello R (ed.) Biomaterials Science and Engineering. Europe, China: InTech, 2011, pp.193–210. Available at: http://cdn.intechopen.com/pdfs/19709/ InTech-Biomaterials_and_biotechnology_schemes_ utilizing_tio2_nanotube_arrays.pdf. 33. Deligianni DD, Katsala ND, Koutsoukos PG, et al. Effect of surface roughness of hydroxyapatite on human bone marrow cell adhesion, proliferation, differentiation and detachment strength. Biomater 2001; 22: 87–96. 34. Yoshinari M, Oda Y, Inoue T, et al. Bone response to calcium-phosphate coated and biphosphonate immobilized titanium implants. Biomater 2002; 23: 2879–2885. 35. Guehennec LL, Soueidan A, Layrolle P, et al. Surface treatments of titanium dental implants for osseointegration. Dental Mater 2007; 23: 844–854. 36. Indira K, Kamachi Mudali U and Rajendran N. Corrosion behavior of electrochemically assembled nanoporous titania for biomedical applications. Ceram Int 2013; 39: 959–967. 37. Truong VK, Lapovok R, Estrin YS, et al. The influence of nano-scale surface roughness on bacterial adhesion to ultrafine-grained titanium. Biomater 2008; 31: 3674–3683. 38. Vasilescu C, Drob P, Vasilescu E, et al. Characterisation and corrosion resistance of the electrodeposited hydroxyapatite and bovine serum albumin/hydroxyapatite films on Ti-6Al-4V-1Zr alloy surface. Corros Sci 2011; 53: 992–999. 39. Park JW, Kim YJ and Jang JH. Enhanced osteoblast response to hydrophilic strontium and or phosphate ions incorporated titanium oxide surfaces. Clin Oral Impl Res 2010; 21: 398–408.
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40. Tang H, Guo Y, Jia D, et al. High bone-like apatiteforming ability of mesoporous titania films. Micropor Mesopor Mat 2010; 131: 366–372. 41. Zeng X, Gan YX, Clark E, et al. Amphiphilic and photocatalytic behaviors of TiO2 nanotube arrays on Ti prepared via electrochemical oxidation. J Alloy Compd 2011; 509: L221–L227. 42. Park JW, Kim HK, Kim YJ, et al. Osteoblast response and osseointegration of a Ti–6Al–4V alloy implant incorporating strontium. Acta Biomater 2010; 6: 2843–2851. 43. Capuccini C, Torricelli P, Sima F, et al. Strontiumsubstituted hydroxyapatite coatings synthesized by pulsed-laser deposition: in vitro osteoblast and osteoclast response. Acta Biomater 2008; 4: 1885–1893. 44. Ni GX, Chiu KY, Lu WW, et al. Strontium-containing hydroxyapatite bioactive bone cement in revision hip arthroplasty. Biomater 2006; 27: 4348–4355. 45. Sila-Asna M, Maeda ABS, Kitaguchi H, et al. Osteoblast differentiation and bone formation gene expression in strontium-inducing bone marrow mesenchymal stem cell. Kobe J Med Sci 2007; 53: 25–35. 46. Nakamura S and Nakahira A. Synthesis and evolution of porous hydroxyapatite prepared by hydrothermal treatment and subsequent sintering method. J Ceram Soc Japan 2008; 116: 42–45. 47. Wei D, Zhou Y, Jia D, et al. Biomimetic apatite deposited on microarc oxidized anatase-based ceramic coating. Ceram Int 2008; 34: 1139–1134.
48. Coreno J and Coreno O. Evaluation of calcium titanate as apatite growth promoter. J Biomed Mater Res Part A 2005; 75A: 478–484. 49. Amin MS, Randeniya LK, Bendavid A, et al. Apatite formation from simulated body fluid on various phases of TiO2 thin films prepared by filtered cathodic vacuum arc deposition. Thin Solid Films 2010; 519: 1300–1306. 50. Kunze J, Muller L, Macak JM, et al. Time–dependent growth of biomimetic apatite on anodic TiO2 nanotubes. Electrochim Acta 2008; 53: 6995–7003. 51. Kim SE, Lim JH, Lee SC, et al. Anodically nanostructured titanium oxides for implant applications. Electrochim Acta 2008; 53: 4846–4851. 52. Karthega M and Rajendran N. Hydrogen peroxide treatment on Ti–6Al–4V alloy: a promising surface modification technique for orthopaedic application. Appl Surf Sci 2010; 256: 2176–2183. 53. Hang R, Huang X, Tian L, et al. Preparation, characterization, corrosion behavior and bioactivity of Ni2O3doped TiO2 nanotubes on NiTi alloy. Electrochim Acta 2012; 70: 382–393. 54. Gad El-Rab SMF, Fadl-allah SA and Montser AA. Improvement in antibacterial properties of Ti by electrodeposition of biomimetic Ca–P apatite coat on anodized titania. Appl Surf Sci 2012; 261: 1–7.
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In-vitro biocompatibility and corrosion resistance of strontium incorporated TiO2 nanotube arrays for orthopaedic applications
Journal of Biomaterials Applications 2014, Vol. 29(1) 113–129 ! The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328213516821 jba.sagepub.com
K Indira1, U Kamachi Mudali2 and N Rajendran1
Abstract This article investigates the in-vitro biocompatibility and corrosion behaviour of strontium ion incorporated TiO2 nanotube arrays formed by anodization method for orthopaedic applications. The morphological studies were carried out using field emission scanning electron microscopy, atomic force microscopy, attenuated total reflectance fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and thin film X-ray diffraction techniques. The morphological investigation indicated that the length and the average diameter of nanotube were 2.1 0.3 mm and 110 4 nm, respectively. The wettability measurements showed that the TiO2 nanotube arrays have super wettability, as well as, strontium ion incorporated TiO2 nanotube arrays exhibited hydrophilic behaviour. Excellent in-vitro bioactivity was observed for TiO2 nanotube arrays with strontium ions. Electrochemical studies in Hank’s solution showed that the TiO2 nanotube arrays with strontium ions have enhanced corrosion resistance. Keywords Anodization, TiO2 nanotubes, dip coating, Hank’s solution, biocompatibility
Introduction Titanium (Ti) and its alloys are widely used as biomedical implant materials owing to their high corrosion resistance, excellent biocompatibility, low specific gravity and long fatigue life.1–6 Basically, metal oxides with nanostructures are capable of improving the longevity of implants, as nanoscale surfaces exhibit uniform surface energy by the interaction with bone cell. For instance, one-dimensional TiO2 nanostructures such as nanotubes and nanowires have appreciable properties such as specific surface area and mechanical strength.7 Thus, TiO2 nanotube arrays (TNTA) have gained considerable attention during the last 15 years.8 Furthermore, nanostructures can be used as a template for the deposition of other materials into the pores, followed by selective etching of the TiO2 mold.9 The TNTA developed posses higher adhesion strength. The TNTA produced in anodization is following selforganization behaviour, thus the tubes are directly attached to the titanium surface and are already electrically connected to the substrate.10 The tubes are growing directly from the titanium substrate, it is considered to be a part of the substrate. Hence, the
adhesion strength of the TNTA is good, which is also reported in the earlier reports.11 In recent times, incorporation of biocompatible elements such as Sr12 and Ag13 into TNTA has triggered enormous attention in biomedical field. Among all, strontium (Sr) has considerable application in biomedical field due to controlled drug delivery system. Oral administration of drug can increase the drug concentration in the blood plasma. On the other hand, a controlled drug delivery system on the biomedical implants can offer a sustained complement of the element or drug. Hence, Sr can be capable of delivering the drug in controlled manner than other elements. Therefore, the release of Sr from the nanostructured surface of Ti implant not only encourages nanosize 1
Department of Chemistry, Anna University, Chennai, India Corrosion Science and Technology Group, Indira Gandhi Center for Atomic Research, Kalpakkam, India 2
Corresponding author: N Rajendran, Department of Chemistry, Anna University, Chennai 600 025, India. Email: [email protected]
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effects but also facilitates in-situ Sr delivery to accelerate osseointegration and favourable osteogenic effects.12 The Sr loaded into TNTA is released to the implant tissue interface locally, which directly enables effective absorption by tissues in the vicinity, thereby promoting bone formation and suppressing bone resorption.14 Hence, the aim of this study is to incorporate Sr ions into TNTA and to investigate the in-vitro and electrochemical behaviour of Sr ion incorporated TNTA (Sr-TNTA) in Hank’s solution. Incorporation of Sr ion was done by simple dip-coating method. The surface morphological, phase structural and topographical properties were observed using microscopic techniques such as field emission scanning electron microscopy (FESEM), thin film X-ray diffraction (TF-XRD) and atomic force microscopy (AFM), respectively. Electrochemical characterization studies were carried out in Hank’s solution using electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization measurements.
Materials and methods Preparation of TNTA by anodization Prior to anodization, the Ti sheets (99.9%, M/s. Ti Anode Fabricators Pvt. Ltd., Chennai, India) were grounded on both sides with silicon carbide paper up to 1000 grit. Upon grinding, the samples were ultrasonically cleaned in a mixture of acetone and 2-propanol for 10 min, then pickled in a mixture of 0.9 M HF and 3.0 M HNO3 for 1 min and dried in air at room temperature. A two-electrode cell set up consisting of platinum cathode of size 1.08 cm3, Ti anode of size 0.11 cm3 were employed for anodization. The anodization was carried out with slight modification in the earlier report.15 The electrolyte used was a mixture of 0.26 M NH4F and 14.5 M ethylene glycol. The constant potential of 50 V was applied for 2.5 h at room temperature with magnetic agitation using DC power supply (M/s Aplab, Model H0615, India). The TNTA was then annealed at 500 C for 2 h. Sr ions were incorporated into TNTA by immersing the anodized specimens in 0.02 M Sr(NO3)2 solution for 10 min and this process was repeated three times. The dip coater used for the present study was Single dip coater (Model SDC 2007C, M/s Apex Instruments & Co., India). The dip-coated specimens were annealed at 500 C for 2 h.
In-vitro studies The in-vitro bioactivity of the specimen was studied by immersing the anodized specimens in Hank’s solution for 7 days and examining the apatite (HAp) forming
ability of the Sr-TNTA. Hank’s solution was prepared as described in the earlier report.3 After immersion in Hank’s solution for 7 days, the specimens were rinsed with distilled water and dried at room temperature. The wettability of the specimens were determined using water droplet contact angle measurement (OCA 15EC, data physics instruments, Germany) with a dosing volume and dosing rate of 10 mL and 1 mL/Sec, respectively.
Surface characterization The surface morphology and elemental composition were characterized by FE-SEM (GEMINI-Supra 55, Germany) equipped with energy dispersive X-ray spectroscopy (EDS). In order to find out the functional groups present on the surface, Attenuated Total Reflectance Fourier Transform Infrared (ATR-IR) spectroscopy was carried out in the frequency range from 400 cm–1 to 4000 cm–1 (Perkin Elmer spectrum 2). The X-ray Photoelectron Spectroscopy (XPS, SPECS made XPS with Al K-a PSOI BOS-150, Germany) was done using Al Ka radiation (1486.71 eV) as the excitation source. The binding energies of target elements (Ti 2 p, O 1 s, Sr 3 d, Ca 2 p and P 2 p) were obtained at the pass energy of 12 eV with the resolution of 0.1 eV. TF-XRD measurements were performed with Bruker D8 Discover (TF-XRD, Germany) using Cu Ka radiation. The surface topography of the samples was seen using AFM (NT-MDT SPM, Russia) in contact mode for a scan area of 20 mm 20 mm and the surface roughness was measured from the AFM topographs using an image analysis tool called ‘NOVA Image Analysis software 1. 0. 26. 1443,’ which is associated with the AFM.
Electrochemical characterization Electrochemical experiments were performed using a conventional three-electrode cell assembly maintained at 37 C. The electrodes namely reference, counter and working electrodes used in the experiments were saturated Ag/AgCl, Pt sheet and test specimens, respectively. Potentiodynamic polarization measurements were carried out for untreated Ti, TNTA and Sr-TNTA after immediate and 7 days of immersion in Hank’s solution using Solartron 1287 Electrochemical Interface. The electrode potential was anodically scanned at a scan rate of 10 mV min–1. All the measurements were carried out in aerated and non-stirred conditions. The corrosion current density (icorr) and corrosion potential (Ecorr) of each sample was calculated from polarization curves with the support of Cview software.
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EIS measurements were carried out using Solartron 1255 frequency response analyzer (FRA) with Solartron 1287 electrochemical interface associated with the standard three-electrode cell assembly as mentioned earlier. All the experiments were carried out in the frequency ranging of 104 to 10–2 Hz, by superimposing an AC voltage of 10 mV amplitude with the data density of 5 points per decade in Hank’s solution. The impedance response obtained over the applied frequency range was represented by Bode plots. For the analysis of impedance results, equivalent circuit was used.16,17
electronic charge transfer, but the gas bubbles were minimum at the end. Figure 2 shows the current transient (I-t) curves recorded during anodization. The initial current was 0.022 mA, which was due to the surface oxidation conditions. The initial higher current drops with time (0–3 s) (inset in Figure 2) indicates the formation of oxide/hydroxide layer over the Ti surface and the subsequent current rise (3–36 s) is due to pitting/ dissolution of the oxide layer by fluoride ions.18 Subsequently, in the next stage, the observed current drop (36–1000 s) indicates the overlap of the above two processes i.e. oxide layer formation and dissolution. At last, the current reaches a steady state which is attributed to the stable film on the surface. It has been reported that, higher current is due to the fast field aided F– transport through the growing oxide layer, which promotes the dissolution of oxide layer.19 Current density is directly related to the tube length, thus, higher current density leads to longer tubes.9
Results and discussion Outlook of the present investigation The schematic representation of HAp growth over Sr-TNTA takes place in three steps and is shown in Figure 1. Initially, anodization of Ti in a mixture of NH4F and ethylene glycol develops the TNTA (first step), in the second step Sr-TNTA layer was formed over the nanotube arrays by dipping in strontium nitrate solution followed by annealing. In the third step, HAp deposits over Sr-TNTA, which has been proved with the support of different studies in the following sections.
Formation mechanism of TNTA During the formation of TNTA, ammonium fluoride and water are ionized into NH4þ, F–, Hþ and OH–, respectively. Ethylene glycol does not participate in the reaction; instead it acts only as a solvent. The possible reaction behind the formation of TNTA is as follows:
Current-time behaviour during anodization
Ti þ 4H2 O
Visible changes were observed at the anode as well as at the cathode during anodization. Different colours form on Ti surface, i.e. from grey to pale green to blue, which indicate the change in the thickness of the oxide layer formed on the surface. Hydrogen gas bubbles evolved throughout anodization at the cathode, which was very intense at the beginning indicating that there was
NH4 F 4 TiðOHÞ4 þ2H2 " HOðCH2 Þ2 OH
Ti þ 4H2 O þ 4NH4 F
" HOðCH2 Þ2 OH 4 TiðOHÞ4 þ4NHþ 4 þ 2F2 " þ2H2
ð2Þ
The F2 and H2 gas evolved at the anode and cathode, respectively. Finally, the product obtained was Ti(OH)4
First step
Second step
TiO2 nanotube arrays (TNTA)
TI substrate
ð1Þ
0.02 M Sr(NO3)2 & Annealing at 500°C for 2 h
Anodization (NH3F + Ethylene glycol)
Srions incorporated TNTA (Sr-TNTA)
Oxide layer Immersion in Hank’s Isolution 7 days TNTA
Sr-TNTA
Sr-TNTA - HAp
Third step
Hydroxyapatite (HA) layer
Figure 1. Schematic illustration of HAp deposited TNTA after the incorporation of Sr ion. TNTA: TiO2 nanotube array.
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0.035 0.034 0.032
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Figure 2. Current-time behaviour of anodized Ti in a mixture of 0.26 M NH4F and 14.5 M ethylene glycol at 50 V for 2.5 h.
and not TiO2. This is in line with the findings of Shin et al.20 Therefore, the following steps were involved in the formation of nanotube arrays: (1) growth of oxide layer on the surface of Ti due to the interaction of O2– and Ti4þ ,21 (2) dissolution of oxide layer, (3) pit formation on the surface and (4) development of pores from the pits.22 In the presence of fluoride ions, the oxide layer partially dissolved along with continuous pit formation and finally, the pits were transformed into pores and simultaneously into tubes. Formation mechanism of Sr-TNTA involves the following reaction (equation (3)); which depicts the precipitation of SrTiO3. The divalent Sr reacts with the titanium hydroxide (Ti(OH)4) and precipitates out SrTiO3. Sr2þ þ TiðOHÞ4 ! SrTiO3 þ H2 O þ H2 "
ð3Þ
Surface characterization The FE-SEM images and EDS results of the specimen are shown in Figure 3(a)–(h). The surface morphology of untreated Ti reveals only the polishing grooves as shown in Figure 3(a). After anodization, nanotubes arrays (Figure 3(c)) with the average diameter and wall thickness of 110 4 nm and 15 2 nm,
respectively, were observed. It is clear from Figure 3(e) that tube structures were formed with a length of approx. 2.1 0.3 mm. From Figure 3(f), it is apparent that the tube mouth was open at the top and closed at the bottom. The average inter-tube diameter (distance between the centre of the adjacent tubes) was found to be 128 2 nm. After dipping the TNTA specimens into strontium nitrate solution, Sr ions cover the walls of the tube. In addition, some Sr ions were scattered like clusters over the top surface of the TNTA. The TNTA (Figure 3(g)) retained the original nanotube structure after the incorporation of Sr ions, however, minor decrease (20 5 nm) in the diameter (during the transformation of TNTA to Sr-TNTA, the diameters of the tubes were changed from 110 to 90 nm respectively) was observed, which was probably due to volume expansion during the transformation from titanium oxide to strontium titanate.14 It has been reported that the inter-pore distance, pore diameter and wall thickness depend linearly on anodization conditions.23 The EDS (Figure 3(b), (d) and (h)) results showed the presence of Ti, O and traces of F on TNTA surface. In addition to Ti and O, Sr is also present on the surface of Sr-TNTA, which confirmed the presence of Sr ions over the TNTA surface. It is reported in the literature that the mechanical properties of the nanostructure Ti is higher than the commercially pure Ti.24 Brammer et al.24 proved that the Ti surfaces with nanotubes interacted most
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Figure 3. FE-SEM images of (a) untreated Ti, (c) TNTA (e) cross sectional view of TNTA and (f) bottom view of TNTA and (g) Sr-TNTA and EDS spectrum of (b) untreated Ti, (d) TNTA and (h) Sr-TNTA.
efficiently with primary bovine chondrocytes by preserving the characteristic spherical morphology and increasing extracellular matrix bril secretion and biochemical production when compared to a plane Ti surface. Park et al.25 reported that the cell adhesion, spreading, and growth on surfaces of this nanometric scale was enhanced in comparison to plain TiO2 surfaces. It is reported elsewhere that the nanoscaled topography can guide enhanced cellular migration on the surface, promote differentiation and matrix production of bone cells and enhance short- and long-term
osseointegration both in vitro and in vivo.14 In the case of nanostructured TNTA, the Sr ions sat on the walls of the tube and bonded to the surface very strongly (since, TNTA has higher adhesion strength than bear Ti), whereas, in the case of plane Ti, the Sr ions were deposited on the compact oxide layer, where the adhesion strength is very poor. Hence, the nanostructured topography could reveal the favorable effect on Sr loading than the plane Ti. Figure 4(a)(d) presents ATR-IR spectra of the samples. The untreated Ti (Figure 4(a)) exhibited a
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% Transmittance
(a)
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O-H
O-O
Sr-O
Sr-O Ti-O
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Figure 4. The ATR-IR results obtained from (a) untreated Ti, (b) as anodized TNTA, (c) annealed TNTA and (d) Sr-TNTA.
weak band at around 440 cm–1, which can ascribe to Ti-O bond originating from the native oxide layer. TNTA (Figure 4(b)) showed a triplet peak from 3448 cm–1 to 2864 cm–1, which are ascribed to v3 and v1 stretching modes of O-H bond (may be due to Ti-OH group).26 The peak at 920 cm–1 is assigned to O-O bond. After annealing (Figure 4(c)), the intensity of O-H bond gets reduced and the Ti-O bond becomes stronger. The Sr-O (Figure 4(d)) bond causes the vibration at 1360 cm–1,27 and the peak at 452 cm–1 is attributed to Ti-O bond. The intensity of the O-H bond diminishes in Sr-TNTA which may due to the annealing effect. ATR-IR confirmed the presence of Sr-O
bond. In addition, the presence of Sr over TNTA surface is further confirmed using XPS and TF-XRD. Chemical state of the elements present on the surface was studied using XPS and is shown in Figure 5(a)–(e). The Ti 2 p signals in TNTA (Figure 5(a)) exhibits two dominant peaks, which corresponds to Ti4þ/TiO2 (Ti 2p1/2 at 465.2 eV and Ti 2p3/2 at 459.4 eV).28 In the O 1 s spectrum (Figure 5(b)), the Ti-OH/Ti-OH2 on TiO2 surface were observed at 530.8 eV and 532.0 eV, respectively.29 The Ti 2p1/2 and Ti 2p3/2 spectrum in Sr-TNTA has the binding energy values of 464.6 eV and 458.6 eV (Figure 5(c)), respectively, which corresponds to SrTiO3.12 The binding energy corresponding to O 1 s
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Intensity, (a.u.)
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(c)
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O1s
Intensity, (a.u.)
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Intensity, (a.u.)
(a)
119
130
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Binding energy (eV)
Figure 5. The XPS spectra of (a, b) TNTA and (c–e) Sr-TNTA.
at 530.0 eV (Figure 5(d)) is attributed to SrTiO3 and 531.3 eV corresponds to Ti-OH group indicating the presence of both TiO2 and SrTiO3 on the surface. The binding energy values at 134.6 and 136.4 eV (Figure 5(e)) were assigned for the Sr 3 d from SrTiO3.30 The crystal structure of as anodized TNTA, annealed TNTA and Sr-TNTA were characterized by TF-XRD and are shown in Figure 6(a)–(c). The diffraction pattern of TNTA shows only the Ti peaks which confirms that the as-anodized TNTA was in amorphous phase.22 The annealed TNTA (Figure 6(b)) exhibited the characteristic diffraction peaks of anatase phase of TiO2 at 2y values of 25.4 and 48.1 (JCPDS No.: 21-1272). The Sr-TNTA were composed of SrTiO3 (JCPDS, card no: 05-0634) and anatase phase of TiO2 (JCPDS, card no: 21-1272), which is in accordance with the XPS results. Anatase peak emerge, implying that part of Ti-O barely reacted with Sr and formed SrTiO3. The rest of the amorphous oxide has not reacted and has been converted into crystalline oxide (anatase) after heat treatment.28
Surface roughness and wettability Several factors such as surface composition, roughness and wettability play a significant role in implant tissueinteraction and osseointegration. Figure 7 shows the bar chart representation of roughness results, which showed that the average surface roughness of untreated
Ti, TNTA and Sr-TNTA were found to be 152, 360 and 125 nm, respectively. The roughness measurements showed that TNTA possess enhanced surface roughness than other samples, which may be attributed to the formation of adherent nanotube layer on Ti surface. The Sr-TNTA has very low roughness compared to the TNTA, which is probably due to the incorporation of Sr ions into the TNTA surface. It has been reported elsewhere that increase in thickness of the anodic layer increases the roughness as well as corrosion resistance of the material.31 Lu et al.9 studied the effect of the surface roughness on the TiO2 nanotubes formation during the anodization, which showed that the nanotubes formed from as-received and chemically polished samples have rough surfaces. As a result, the corresponding nanotube morphologies and the bottom oxide barrier layer are non-uniform. Generally, for bio-active implant materials, enhanced surface roughness is one of the most important key factors to provide better cell response. It has been reported that, besides surface chemistry and crystallinity, surface roughness influences the cellular response, enhancing cell adhesion and proliferation.32 Lower surface roughness gives a lesser bone contact,33 and the higher surface roughness provides not only an early fixation but also better mechanical stability for implants, which would result in good mechanical interlocking between the implant surface and bone ingrowth.34 The surface roughness of 10 nm to 10 mm
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(c)
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Figure 6. The TF-XRD patterns of (a) as anodized TNTA, (b) annealed TNTA and (c) Sr-TNTA.
350
Average roughness, nm
300 250 200 150 100 50 0 Untreated Ti
TNTA
Sr-TNTA
Figure 7. Bar diagram representing the results of surface roughness. TNTA: TiO2 nanotube array.
can induce the apatite growth, thus improving the bioactivity of the material.35,36 It is reported elsewhere that the surface architecture at nanoscale may be one of the major factors influencing bacterial cell attachment to the surfaces.37 It is conceivable that the film with high roughness would have more adsorption sites of hydroxyls on the TiO2 surface, thus, the hydroxyl groups
combines with the positively charged calcium ions, which in turn combines with phosphate ions and form Hap.38 Park et al.39 reported that Sr incorporation into titanium oxide layer further enhance the cell attachment, feasibility of hydrophilic and micro-rough Ti surface. In order to achieve favourable implant bone healing, micro-rough surface property is one of the desirable factors. Hence, from the roughness value of Sr-TNTA, it is clear that it has mirco-rough surface, which is considered potentially favourable for implant materials. Contact angle is indirectly related to surface energy. Lower the contact angle, higher will be the surface energy.40 Higher surface energy materials favour the formation of HAp in biological solution. Super wettability of the specimens, with high surface energy, can induce HAp formation. Figure 8 shows the pictorial representation of the contact angle measurements of the specimens. The contact angle for the untreated Ti was 86 . Super wettability was observed for TNTA whereas the contact angle for Sr-TNTA was found to be 26 , which may be due to the presence of partial TiO2 on their surface. The hydrophilic nature of the TNTA was attributed to the capillary effect of the nanotubes.41 Improved wettability i.e. low contact
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Contact angle, (Degree)
100
80
In-vitro characterization 60
40
20
0 Untreated Ti
TNTA
Sr-TNTA
Figure 8. Pictorial representation of contact angle results of the specimens. TNTA: TiO2 nanotube array.
(a)
In order to assess the biocompatibility, the specimens were immersed in Hank’s solution for 7 days. Figure 9(a)–(i) displays the FE-SEM morphology and EDS spectra of untreated Ti, TNTA and Sr-TNTA after immersion in Hank’s solution for 7 days. Cluster-like particles were observed to be non-uniformly deposited over the untreated Ti surface (Figure 9(a) and (b)) after 7 days of immersion in Hank’s solution. Globular particles covered the TNTA (Figure 9(d) and (e)) layer whereas in the Sr-TNTA surface trabecular bone like morphology was observed uniformly all over the
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Figure 9. FE-SEM micrographs (a, d and g (low magnification views) b, e and h (high magnification views)) and EDS spectra (c, f and i) of untreated Ti, TNTA and Sr-TNTA specimens after immersion in Hank’s solution for 7 days.
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surface (Figure 9(g) and (h)). The EDS results (Figure 9(c), (f) and (i)) confirmed the presence of Ca and P over the surface (main elements in HAp), which correlated well with the results of ATR-IR, XPS and TF-XRD. From the images it is obvious that the HAp deposition was observed not only in a small part of the SrTNTA surface but in the whole area of the sample. However, Sr-TNTA is less hydrophilic than TNTA which may due to the higher crystalline nature of the Sr-TNTA (observed from the TF-XRD pattern). Generally, the hydrophilic surface (contact angle less than 90) can provide better bioactivity.36 It is reported elsewhere that the Sr is a potent bioactive element in the enhancement of bone healing, hence, the incorporation of Sr incorporation promoted the attachment and proliferation of osteoblastic cells on calcium phosphate bone substitutes and HAp coatings in vitro.42
Capuccini and co-workers reported the substitution of Sr on HA coating towards the in vitro osteoblast and osteoclast response. They found that the presence of Sr in the coating enhances osteoblast activity and differentiation, while it inhibits osteoclast production and proliferation.43 Also, it is reported by Ni et al.,44 that Sr-containing HA bone cements reduced the possibility of wear debris at the bone–cement interface, stimulated new bone formation and better physiological stress transfer between the implant and host bone in vivo. Osteoblastic differentiation from human mesenchymal stem cell (hMSCs) is an important step of bone formation. In order to study the effect of Sr on hMSCs and osteoblastic lineage, Sila-Asna et al. has studied the in vitro induction of hMSCs by using strontium ranelate, a natural trace amount in water, food and human skeleton. They conclude from their studies that the Sr is an important factor for inducing mesenchymal stem
% Transmittance
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Figure 10. The ATR-IR results for (a) untreated Ti, (b) TNTA and (c) Sr-TNTA after immersion in Hank’s solution for 7 days.
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ð4Þ
The negatively charged sites present on the TiO2 surface attract the positively charged Ca2þ, thus, promoting the nucleation of HAp. In order to evaluate the oxidation state of the specimen after immersion in Hank’s solution for 7 days, XPS study was carried out for TNTA and Sr-TNTA, which is presented in Figure 11(a)–(f). The O 1 s spectrum of TNTA at 532.4 eV is deconvoluted into two peaks arising from C-O and/or hydroxyl group and 534.1 eV from phosphate group. The binding energy values of Ca 2 p (Figure 11(b)) are at 348.6 eV and 352.2 eV, which is attributed to calcium hydrogen phosphate. The P 2 p (Figure 11(c)) peak at 134.1 eV is from phosphate group. The O 1 s spectrum (Figure 11(d)) of Sr-TNTA is deconvoluted into two peaks with the binding energy of 530.7 eV and 533.3 eV, which corresponds to oxygen in phosphate (PO43–) and hydroxyl group, respectively.49 The Ca 2 p spectra of Sr-TNTA
348.6
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Intensity, (a.u.)
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was also found into the HAp layer. A study by Coreno et al.48 showed that the formation mechanism of HAp layer over crystalline SrTiO3 is similar to that of crystalline CaTiO3. SrTiO3 acts partly as insoluble TiO2 and partly as soluble SrO. The equation (4) describes the subtle dissolution of SrTiO3:
Intensity, (a.u.)
cells to differentiate into osteoblasts with further enhancement on bone formation. In addition, they show strong evidence of bone structure stabilization by expressing genes related to bone formation at early day of cell differentiation.45 Thus, incorporation of Sr into TNTA is a successful approach to enhance the osteoconductivity of implant materials. ATR-IR spectra of untreated Ti, TNTA and SrTNTA on immersion in Hank’s solution for 7 days are shown in Figure 10(a)–(c). The peaks at around 799 and 735 cm–1 for untreated Ti are attributed to the presence of P2O74– (pyrophosphate) group.46 Characteristic peak of v3 PO43– group was observed at 1030 cm–1 for untreated Ti. In the TNTA and SrTNTA spectra, a broad absorption band around 3680 to 2690 cm–1 originates from the v3 and v1 stretching modes of O-H bond, and the band at 1650 cm–1 is ascribed to v2 bending mode of O-H bond. A small band at 1420 cm–1 arises from CO32– group and a strong band at 1032 cm–1 is attributed to v3 mode of PO43– group.47 The bands at 658, 596 and 554 cm–1 are attributed to the crystallized HAp layer. The peaks corresponding to the phosphates and carbonates are identified on the ATR-IR spectra, which confirmed the formation of HAp over the Sr-TNTA surface; which is further proved by XPS and TFXRD studies. However, trace amount of carbonate
344 346 348 350 352 354 356
Binding energy (eV)
Sr 3d/P 2p
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130
132
134
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Figure 11. The XPS spectra of (a–c) TNTA and (d–f) Sr-TNTA after immersion in Hank’s solution for 7 days.
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Ti TiO2 SrTiO3 HAP
Intensity, (a.u.)
(b)
(c)
(d)
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2 theta , (degree)
Figure 12. The TF-XRD patterns of TNTA and Sr-TNTA after immersion in Hank’s solution for 7 days: before heat treatment (a and b) and after heat treatment (c and d).
had the binding energy of 348.2 and 351.9 eV, which is ascribed to calcium phosphate.50 Sr 3 d and P 2 p (Figure 11(f)) are co-analyzed because of the overlap between the two (Sr 3 d and P 2 p). The binding energy value at 134.9 eV is attributed to Sr 3d3/2 and the binding energy value of 133.7 eV is assigned to phosphorous in PO43–.51 Hence, the XPS study confirms that Ca, P and Sr containing phases (Ca3PO4 and SrTiO3) exist on the nanotube surface. Figure 12(a)–(d) represents the TF-XRD pattern of TNTA and Sr-TNTA after immersion in Hank’s solution for 7 days. The characteristic 2y peaks of HAp observed at 28.4 , 31.7 , 32.9 , 42.1 , 46.7 and 49.1 correspond to (102), (211), (300), (311), (222) and (213) planes of TNTA. The TF-XRD spectrum of Sr-TNTA discloses the existence of HAp layer (JCPDS, card
no: 09-0432) on crystalline Sr-TNTA. After heat treatment (Figure 12(c) and (d)) the crystalline HAp peaks at 25.9 corresponding to (002) plane is clearly visible and all the peaks become sharper than the previous one. These results together with ATR-IR and XPS confirmed the growth of HAp over the surface.
Electrochemical characterization The potentiodynamic polarization curves of untreated Ti, TNTA, Sr-TNTA after immediate and 7 days of immersion in Hank’s solution are presented in Figure 13(a) and (b). The current density remained almost constant and extended over a wide range of potential for all the samples. Insignificant increase in current density after 2 V was observed for untreated Ti and TNTA,
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(a) 3
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10–4
10–8
10–7 10–6 Current density, i/Acm–2
10–5
Figure 13. Potentiodynamic polarization results of untreated Ti, TNTA and Sr-TNTA after immersion in Hank’s solution: (a) immediate immersion and (b) 7 days immersion. TNTA: TiO2 nanotube array.
Untreated Ti TNTA Sr-TNTA Simulated
–Phase angle, degree
80
60
40
(b)
Log / Z /,0hm cm2
(a)
104
103
102
20
101
0
(c)
103
104
Untreated Ti TNTA Sr-TNTA Simulated
80
60
40
20
10–2 10–1 100 101 Log f, Hz
102
(d)
103
104
Untreated Ti TNTA Sr-TNTA Simulated
105 Log / Z /,0hm cm2
10–2 10–1 100 101 102 Log f, Hz
–Phase angle, degree
Untreated Ti TNTA Sr-TNTA Simulated
105
104
103
102
0 10–2 10–1 100
101 102 Log f, Hz
103
104
10–2 10–1 100
101 102 Log f, Hz
103
104
Figure 14. The EIS results of untreated Ti, TNTA, Sr-TNTA specimens after immersion in Hank’s solution: (a and b) immediate immersion and (c and d) 7 days immersion. TNTA: TiO2 nanotube array.
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Rs
Rb Qb
(b)
Rs
Rp
Rb
Qp
Qb
(c) Rs
Ra
Rb
Qa
Qb
(d) Rs
Ra
Rp
Qa
Qp
Rb Qb
Figure 15. The equivalent circuit models of the specimens after immersion in Hank’s solution: (a) untreated Ti after immediate immersion, (b) TNTA and Sr-TNTA after immediate immersion and (c) untreated Ti after 7 days of immersion and (d) TNTA and SrTNTA after 7 days of immersion.
which may be due to electron transfer across the oxide layer.1 The current density was minimum (Figure 13(b)) for Sr-TNTA after 7 days of immersion in Hank’s solution, which is due to the formation of stable HAp layer over Sr-TNTA surface, which was already confirmed by FE-SEM-EDS, ATR-IR, XPS and TF-XRD studies. An obvious increase in current between 1 V and 2 V was observed for Sr-TNTA after immersion in Hank’s solution and also the current density is higher after 1.7 V compared to TNTA sample. This can be attributed to the structural changes of the passive layer or changes in the ionic or electrical conductivity of the layer.52 Figure 14(a)–(d) shows the EIS for untreated Ti, TNTA, Sr-TNTA after immediate and 7 days of immersion in Hank’s solution. The Bode-phase angle maximum for TNTA and Sr-TNTA at lower frequency region was –57 and –56 and higher frequency region was –58 and –34 , respectively. After 7 days of immersion, the phase angle plot for untreated Ti exhibited two distinct behaviours and attained a maximum value of –77 at the intermediate frequency region. The phase angle for the lowest frequency was –41 . Significant shift in the phase angle for Sr-TNTA specimens after immersion in Hank’s solution for 7 days showed three distinct phase angle behaviour at higher, intermediate and lower frequency regions compared to other specimens. The phase angle values were found to
be –10 and –65 at higher and lower frequency regions, respectively. The equivalent circuit models proposed to obtain the best fitted results are shown in Figure 15(a)–(d). After immediate immersion, untreated Ti showed single time constant whereas, TNTA and Sr-TNTA showed two times constants, which is attributed to the inner barrier layer and outer porous layer. Untreated Ti after 7 days of immersion in Hank’s solution showed two time constants, whereas the TNTA and Sr-TNTA after immersion in Hank’s solution for 7 days showed three time constants. It has been reported that nucleation and growth of bonelike apatite takes place on untreated Ti surface on prolonged immersion in SBF solution.52 The third time constant in the TNTA and Sr-TNTA after immersion may be due to the newly formed HAp layer, which has been confirmed by EDS, ATR-IR, XPS and TF-XRD characterization. The calculated impedance parameters are given in Table 1. It was observed that the Rb of the anodized samples (TNTA, Sr-TNTA and Sr-TNTA after immersion in Hank’s solution for 7 days) showed enhanced value than untreated Ti, which confirms that the anodization process increases the thickness of the barrier layer. The Rp value of Sr-TNTA was higher compared to TNTA, which indicated that the increased thickness of the outer layer is due to the incorporation of Sr ions
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Table 1. Electrochemical impedance spectroscopy (EIS) parameters of untreated Ti, TNTA and Sr-TNTA after immediate and 7 days of immersion in Hank’s solution. Specimen Immediate immersion Untreated Ti TNTA Sr-TNTA 7 days immersion Untreated Ti TNTA Sr-TNTA
Rs (k cm2)
Ra (k cm2)
Qa (Fcm–2Sn)
na
Rp (k cm2)
Qp (Fcm–2Sn)
np
Rb (k cm2)
Qb (Fcm–2Sn)
nb
0.01 0.06 0.02
– – –
– – –
– – –
– 3.64 6.25
– 8.95 10–7 4.10 10–7
– 0.81 0.88
225 1410 1350
4.20 10–5 2.40 10–6 1.24 10–6
0.92 0.82 0.89
0.04 0.01 0.03
0.02 9.10 15.60
1.71 10–7 1.39 10–7 1.45 10–7
0.83 0.80 0.86
– 5.28 6.94
– 6.10 10––7 3.16 10–7
– 0.81 0.89
550 1248 1235
4.16 10–6 1.76 10–6 1.44 10–6
0.87 0.85 0.83
Rs: solution resistance; Rb: polarization resistance of barrier layer; Qb: double layer capacitancee of barrier layer; Rp: polarization resistance of porous layer; Qp: double layer capacitance of porous layer; Ra: polarization resistance of HAp layer and Qa: double layer capacitance of HAp layer; TNTA: TiO2 nanotube array.
over the TNTA surface. The pathways for ion transportation were offered by rough and porous layer, which decreases their barrier functions.53 Significant increase of porous layer resistance after immersion in Hank’s solution for 7 days suggests excellent HAp growth, which in turn is related to better corrosion resistance of the material. High Rp values indicated high compactness and passivity of the surfaces. The Ra is higher for Sr-TNTA meaning that thick HAp layer was formed over the surface. Lower Qp value for Sr-TNTA after 7 days of immersion is due the wadding of nanotube surface by the newly grown HAp layer.54 The ‘n’ values of all the layers were close to 1 which means all the layers behaved as an ideal capacitor. Hence, Sr-TNTA is an interesting material which offers the required bio-compatibility in the body fluid, making it applicable for orthopaedic implants.
Conclusions In summary, Sr-TNTA was successfully fabricated on Ti substrate by dip-coating method of anodic TNTA. The nanotube morphology is retained with small reduction in the tube diameter after the complete incorporation of Sr ions over the TNTA tube walls. HAp grown Sr-TNTA is considered potentially encouraging material for implant application, which is evident from the roughness and contact angle results. In vitro experiments demonstrated that Sr-TNTA can accelerate HAp growth in Hank’s solution; electrochemical experiments indicated good corrosion resistance and biocompatibility of the Sr-TNTA. Hence, it can be concluded that Sr-TNTA is a viable alternative in orthopaedic applications to provide improved corrosion resistance and enhanced biocompatibility.
Acknowledgements The authors acknowledge the University Grants Commission - Department of Atomic Energy - Consortium for Scientific Research for funding this project. The authors also thank Dr. M. Kamruddin and Ms. Sunitha, Materials Science Group, Indira Gandhi Centre for Atomic Research, Kalpakkam for their assistance in FE-SEM and TF-XRD characterization respectively. DST-FIST and UGC-DRS are gratefully acknowledged.
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