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Original Article
DOI: 10.1016/j.jmrt.2019.09.049
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Available online 11 October 2019
Post treatments effect on TiZr nanostructures fabricated via anodizing
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Maria Vardakia,b, Shiva Mohajerniab, Aida Pantazic, Ionela Cristina Nicad, Marius Enachescuc, Anca Mazareb, Ioana Demetrescua,e,
, Patrik Schmukib,**
a General Chemistry Department, University POLITEHNICA of Bucharest, Str. Polizu 1-7, 011061, Bucharest, Romania
b WW4-LKO, Department of Materials Science, Friedrich Alexander University of Erlangen Nurnberg, Martensstr. 7, 91058, Erlangen, Germany
c Center for Surface Science and Nanotechnology University POLITEHNICA of Bucharest, Splaiul Independentei nr. 313, 060042, Bucharest, Romania
d Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Bucharest, Splaiul Independentei 91-95, 050095, Bucharest, Romania
e Academy of Romanian Scientists, Splaiul Independentei 54, 050094 Bucharest, Romania
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Table 1. Chemical formula, crystal system, and cell parameter of orientation sample anodized, anodized and air annealed and anodized, air annealed and reduced.
Table 2. Roughness parameters, RMS and Ra, determined from 2 × 2 μm2 topography scans.
Table 3. Contact Angle (o), Roughness (RMS and Ra) and mean adhesion force values obtained for 1 × 1 μm2 topography scan.
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Abstract

The primary focus of the paper is the fabrication of nanotubes via a two steps anodizing procedure on a TiZr substrate followed subsequent annealing treatments and their effect on surface and biocompatibility. The two step anodizing process is optimized for obtaining uniform and ordered nanotubular structures, that are then annealed in air or reduced in an Ar/H2 environment. The high aspect ratio nanotubular structures (diameter 160 nm, length 9.4 μm) maintained their morphology after annealing or reduction. While the thermal treatments burned-off the fluoride, they converted the tubes to ZrTiO4 (annealing in air) or (Zr0.333 Ti0.666)O2 (reducing). Surface roughness and adhesion forces evaluation of the nanostructures was included in the AFM investigations, and show that these properties can be tuned. In vitro cell response to TiZr substrate and nanotubular coatings were analyzed using HGF-1 gingival fibroblasts cell line and LDH, nitric oxide (NO) and reactive oxygen species (ROS) tests. Regarding the hydrophilic balance of the studied samples, as expected it decreased after annealing and but increased after reduction, with only small change in the cell response. The results suggest that these coatings are biocompatible with human gingival fibroblast and can be used in biomedical applications without generating any considerable inflammatory process.

Keywords:
Anodizing
Thermal treatments
Roughness
Adhesion
Biocompatibility
Full Text
1Introduction

Metallic materials offer development and new breakthrough for technological and practical applications [1–5]. Their remarkable properties are a synergetic achievement of the combined engineering of many factors including alloy composition, surface properties, microstructure, grain size, and others. In the development of surface and coating technology [6,7], the goal is not only to improve properties of the existing materials but also to create a new generation of materials, where the properties are being designed as to fulfill specific demands of different fields such as energy [8], corrosion protection [9], and bioapplications [10,11]. Due to the aggressive bacteria nowadays, a great number of researches were devoted in the last decade to investigating the antibacterial activity [12–14]. Bacteria growth is one of the failures of metallic implants after diverse surgery procedures and various drugs [15] were recently proposed for tailoring antibacterial, biocompatibility and stability properties in the exploitation time of the implant life. In fact, stability, biocompatibility and antibacterial properties are important features of all biomaterials used in biomedical applications including all kind of metallic materials for dental work [16] or for other implants [17].

Among other metallic biomaterials surgical stainless steel (316), CoCr and Ti based alloys are most frequently used; as well as more recently biodegradable Mg alloys are also widely investigated [18]. Alloying of Ti with Al, Nb, Ta, Zr, V was proposed in order to enhance the mechanical properties and led to binary or ternary alloys [19,20]. As valve metals which form a passive oxide stable and able to induce an improved cell response and osseointegration, all such possibilities were proposed and tested. Modern implant dentistry introduced zirconia and Strauman Roxolid [20] as titanium-zirconium alloys with 13–17% zirconium (TiZr1317) which are gaining popularity and are being considered as a first treatment option dominating over the other treatment modalities.

ZrTi alloys are currently explored in biomedical applications due to the exceptional biocompatibility of both Ti and Zr combined with their enhanced properties, such as superior tensile and fatigue strength compared to pure Ti [21] and better mechanical properties compared to pure Zr [22]. These properties make TiZr alloys ideal candidates for medical applications [23,24].

A crucial aspect for the use of TiZr alloys in biomedical applications, as well as for other Ti or Ti based alloys, is the nanostructuring of the surface as the nanoscale morphology affects cells interaction. A most elegant method to achieve this is by self-ordered nanotube arrays that can be grown on such substrates by electrochemical anodizing. These structures provide the advantage of nanotubular oxide morphology with controllable dimensions (diameter, length) perpendicularly aligned on the substrate, and different morphologies can be obtained by changing the anodizing conditions (anodizing time, anodizing potential, temperature, steps of anodizing etc.) [25–29].

Nanostructures grown on TiZr alloy show a partially crystalline structure, depending on anodizing conditions, composition of the alloy (percentage of Zr) or the subsequent annealing treatment [23,30–32]. To note that nanostructures grown on Zr substrates show a crystalline structure [33–35], while those on pure Ti usually are amorphous [36,37]. Both constituent elements of the ZrTi alloy contribute to the composition of the final nanostructure morphology, i.e., the competition between the oxide formation and the chemical dissolution of the oxide induces the development of binary or even multicomponent oxides nanostructures [38].

The morphology of the nanostructures influences the electrochemical stability, wettability and biocompatibility of samples [40,41], and it can be easily tailored by the anodizing conditions [25,38,39]. Moreover, the desired wettability and surface roughness can be further achieved by changing one or more of the anodizing conditions, i.e., electrolyte, applied potential, time etc. [42,43].

Within the present paper we show the influence of the second anodizing on the morphology of nanotubes grown on TiZr (50%) by a two-step anodizing process. Moreover, we evaluate the effect of the calcination and reduction treatment on the structure, biocompatibility and adhesion properties of the tubes. We further evaluate the cell behavior on anodized nanotubes grown on ZrTi alloy and we observe an improvement of the cell response on these structures.

2Experimental2.1Sample preparation

The bulk material composition is TiZr with 50% Zr and 50%Ti (from ATI Wah Chang Co). Prior to anodizing, all studied samples were grinded with abrasive SiC paper up to 1200 grit, cleaned in an ultrasound bath with acetone, ethanol, distilled water for 5 min each and dried with N2 stream. The formation of nanotubes via anodizing of TiZr substrates was carried out in a typical two electrodes electrochemical cell in two steps with Pt used as counter electrode and the TiZr used as working electrode.

The two steps anodizing took place in a Glycerol 15 vol% H2O + 0.2 M NH4F electrolyte. The duration of the first anodizing step was 4 h at 55 V. After the first step, the samples were sonicated in water resulting in the removal of the formed oxide layer. By this way a prepatterned surface was obtained from the first anodizing. Afterwards, the patterned samples were used as substrate in the second step of anodizing. The second step was performed in the same electrolyte at 75 V for 1 h. Immediately after the second step of anodizing the samples were immersed in ethanol for 20 min, washed thoroughly with distilled water and finally dried with N2 stream. Subsequently the samples were annealed at 450 °C for 1 h in air using a tube furnace. The reduced nanotubes were achieved by annealing the samples in Ar/H2 10% at 600 °C for 1 h.

2.2Sample characterization

Samples' morphologies were investigated using a scanning electron microscope (SEM) Hitachi FE-SEM 4800. The FE-SEM is equipped with an energy dispersive X-ray (EDX) setup.

X-ray diffraction measurements were carried out in the 5–100° range on a Rigaku Smart Lab X Ray Diffractometer (Rigaku Corporation, Tokyo, Japan) using Cu Kα radiation (λ = 0.154060 nm) operating at room temperature. The identification of the phase was made by referring to the International Center for Diffraction Data – ICDD (PDF-2) database.

The composition and the chemical states were characterized using X-ray photoelectron spectroscopy (XPS, PHI 5600, US), and peak positions were calibrated on the Ti2p peak at 458 eV.

Topography, roughness and adhesion studies were carried out using a multimode commercial atomic force microscope (Solver Next – NT-MDT), in ambient conditions. Cone-shaped tips from monocrystalline silicon (tip radius ∼10 nm) on cantilevers with stiffness of about 0.65 N/m were used to perform the measurements. The Root Mean Square roughness (RMS) and the Ra parameters were calculated from the acquired topographic images via an image processing software, using the following equations. The root-mean-square roughness – RMS is defined as:

where hi, represents the height value at each data point, h¯ represents the profile mean value of the surface, and N represents the number of data points in the analyzed profile. The arithmetic average roughness-height Ra represents the arithmetic means of the deviations in height from the profile mean value:

The adhesion force variations mappings were acquired in the same area on the sample surface where the topography images were recorded (1 × 1 μm2 scan size), each of them summing 100 curves. All the force-distance curves were measured applying a constant force between the tip and the sample. The adhesion force (F) between the tip and the surface is calculated as F = k × Δd (Hook’s law), where k is the cantilever spring constant and Δd is the deflection distance. The variation of adhesion force is also dependent on contact area, while the contact area is related to the sample and tip geometry.

The hydrophilic/hydrophobic balance was evaluated from contact angles measurements using a drop shape analyzer — DSA 30 equipment. All the measurements were performed with distilled water. Each contact angle value is the average of 10 measurements.

2.3In vitro biocompatibility assessment

In vitro cell response to uncoated TiZr substrate and nanotubes-based coatings were analyzed using HGF-1 gingival fibroblasts cell line (purchased from American Type Culture Collection (ATCC), Cat. No. CRL-2014, Rockville, MD, USA). The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco/Invitrogen, Carlsbad, CA, USA) with an addition of 10% fetal bovine serum (FBS; Gibco/Invitrogen, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere with 5% CO2. After 24, 48 and 72 h of cell exposure to the sterilized samples, several biocompatibility tests were performed. The cell viability was measured using a lactate dehydrogenase (LDH) assay kit (Cytotoxicity Detection Kit-LDH, Roche, Basel, Switzerland) as a measure of cell membrane integrity by reading the absorbance at 490 nm using a FlexStation 3 microplate reader (Molecular Devices, USA). In addition, the level of nitric oxide (NO) released in the culture medium as an indicator of inflammation was assessed using the Griess reagent (a stoichiometric solution (v/v) of 0.1% naphthylethylendiamine dihydrochloride and 1% sulphanilamide in 5% H3PO4) after reading the absorbance at 550 nm. The intracellular ROS level was assessed using a fluorescent compound 2′,7′-dichlorofluorescein diacetate (DCFH-DA, Sigma-Aldrich, St. Louis, MO, USA). The fibroblasts were washed with PBS and incubated with the dye for 30 min at 37 °C. Afterwards, the excess dye was removed and the cells were resuspended in PBS and detached by scraping. The fluorescence was quantified using a fluorimeter (FlexStation 3, Molecular Devices, USA) (excitation wavelength = 488 nm and emission wavelength = 515 nm).

2.4Statistical analysis

All data were expressed as mean value ± SD of three independent experiments. Statistical differences between samples and control were evaluated by Student’s t-test (Microsoft Excel) and a value of p < 0.05 was considered statistically significant.

3Results and discussion

Fig. 1a,b shows the SEM images of top and cross-section of the Ti50%Zr oxide nanotubes obtained via a two step anodizing, namely the nanotubes obtained in the first step are removed by ultrasonication and the prepatterned surface is used as substrate in the second step (for more details please see sample preparation). The resulting nanotubular morphology is indicated for both cases, first when the same anodizing potential is used in both steps and when a different different potential is used (this represent the optimized condition).

Fig. 1.

Top view and cross section images of nanotubes obtained by: a) a two step anodization using similar anodizing voltage, b) optimized condition using different voltages, with the corresponding thermal treatments c) annealing, d) reducing. EDX spectra of e) annealed and f) reduced nanotubes.

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The SEM images clearly show that a highly ordered and well-defined nanotubular structure with a diameter of 160 nm and length of 9.4 μm was successfully obtained by anodizing TiZr alloy in the optimized condition, showing also an open top morphology without the presence of an initiation layer (as observed in Fig. 1a — where such an initiation layer is present at top and in each tube/imprint from the first step there are 2–4 smaller pores). A two step anodizing procedure is generally employed in order to improve the nanotube order and when the voltage in the second anodizing step was increased from 45 V to 75 V, to ensure that nanotubes with the same diameter as that of the dimples are formed. As evident from the SEM images, the open tube diameter is well matched in a 1:1 ratio with the dimple. It is also worth mentioning that both air annealing and the reduction treatment did not have a significant influence on the structure, and that the tubes maintained their initial morphology (except for a slight decrease of the inner diameter of the tube), as shown in Fig. 1c,d. Some small thermal microcracks are observed on the surface of the nanotube samples after annealing in air, but this usually occurs during annealing and reduction of nanotubes grown in organic electrolytes [44] and to a lesser extent for tubes grown in aqueous containing electrolytes [45]. Additionally, the elemental composition of the nanotubular structures was computed from the EDX results (Fig. 1e,f). The data confirms the presence of Ti, Zr and O in all samples. To note, that the as formed anodized sample (i.e. without any other post treatment) has a small amount of F (see supporting information, Fig. S1), this is residue from the anodizing electrolyte and from the fluoride rich layer present in the tubes. All samples present a larger amount of Zr compared to Ti, and the ratio between their sum and oxygen is almost 2 corroborating the presence of oxides.

To further characterize the crystal structure, the measured XRD patterns are shown in Fig. 2a for the as formed, annealed and reduced nanotubular structures (the spectra for the TiZr substrate is shown in Fig. S2).

Fig. 2.

a) XRD patterns of the as formed, annealed and reduced nanotubes. XPS analysis showing the C1s (b), O1s (c), Ti2p (d) and Zr3d (e) peaks of annealed and reduced nanotubes grown on TiZr alloy.

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As it can be seen in the XRD diffraction patterns, we observe that the as-formed anodized sample has a hexagonal crystal system with a (101) orientation, while the annealed or reduced samples have an orthorhombic one with a (111) orientation. Also, with air annealing an increase in volume and cell parameters is observed (Table 1).

Table 1.

Chemical formula, crystal system, and cell parameter of orientation sample anodized, anodized and air annealed and anodized, air annealed and reduced.

Sample  Chemical formula  Orientation  Crystal system  Volume  Cell parameters 
NTs  Ti0.67 Zr0.33  (101)  Hexagonal  39.171  a = 3.0510 b = 3.0510 c = 4.8590 
(ann) NTs  ZrTiO4  (111)  Orthorhombic  132.690  a = 5.0358 b = 5.4874 c = 4.8018 
(red) NTs  (Zr0.333Ti0.666)O2  (111)  Orthorhombic  129.328  a = 4.7112 b = 5.4944 c = 4.9962 

The oxygen resulted from the anodizing process is amorphous and is found in the first region of the difractogram (Fig. 2a). This might suggest that the oxygen is only found at the surface of the nanotubes, the core being composed only of the intermetallic TiZr. By annealing in air the tubes are converted to srilankite (ZrTiO4), which by the reducing treatment is converted to a less oxygen rich form.

In Fig. 2b–e the obtained spectra from the XPS analysis are shown, evaluating the formed nanotubes after the air annealing treatment and after the reduction treatment. It can be observed that besides the distinct differences in the C1s spectra of air annealed samples with and without the reduction treatment, the rest of the spectrum (Ti2p, O1s, Zr3d) are in a satisfactory agreement. The Ti2p peaks have binding energies around 458 eV, while the Zr3d present binding energies at 182 eV. These binding energies are typical of the fully oxidized zirconium ion in its Zr4+ and titanium ion in its Ti4+ state [45,46]. Moreover, the Ti2p XPS spectra of both types of samples are fitted with two main peaks, which are both attributed to titanium ion Ti4+, indicating the existence of TiO2[45,47]. To note that considering the measured C1s peaks, peaks were calibrated to Ti2p (at 458 eV), and no significant difference (broadening) of the Ti2p peaks were observed, that could be related to the presence of Ti3+ states (resulting from the Ar/H2 reduction treatment). This, as even though such treatments promote the formation of Ti3+ centers [44], they cannot be confirmed by XPS as at the surface they are easily oxidized back to Ti4+ when exposed to ambient air. The phase transformation during the air annealing and/or reduction treatment, as well as the different coordination of the oxide ions [48] result in a minor splitting of the O1s spectrum with binding energy around 532 eV, indicating the presence of the ZrTiO4[46]. Such results are consistent with the XRD data that indicated as well the presence of the ZrTiO4 phase.

The AFM topography measurements were successfully carried out at two different scan sizes (2 × 2 um2 and 1 × 1 um2) in two operational modes [49], tapping AFM in which height (topography) and phase signals were acquired simultaneously, and contact AFM in which the adhesion force distribution was studied. The AFM topography and phase images, displayed in Fig. 3, were acquired in tapping mode for all samples, in order to examine the calcination and reduction effect on the surface morphologies. Phase images can provide additional information about surface characteristics, which are sometimes obscured in topographic maps.

Fig. 3.

2D AFM surface topography and phase images on 2 × 2 μm2 for: a, b – as formed, c, d – annealed and e,f– reduced nanotubes.

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The topography images confirm the surface features with hollow nanotubular structures formed as a result of the electrochemical anodizing process. According to the AFM results, both thermal treatments i.e. calcination and reduction (applied in order to crystallize the nanotubes) have an influence on their surface morphologies. A slight decrease in the inner diameter of the ZrTi-oxide nanotubes together with an increase in the dimension of nanotubes’ walls is observed. This tendency of increasing the tube wall thickness could be due to the high annealing temperature. However, the hollow nanotubular configuration is still clearly observed for both the annealed and reduced samples.

Additionally, the roughness parameters calculated from 2 × 2 μm2 topographic images (listed in Table 2) are also showing the changes in morphology occurring after the treatments. Namely, the roughness parameter values have significantly decreased after applying the annealing or the reduction treatment (however, there are no noteworthy differences in the morphological properties provided by the reduction treatment).

Table 2.

Roughness parameters, RMS and Ra, determined from 2 × 2 μm2 topography scans.

Sample  RMS (nm)  Ra (nm)  Skewness 
NTs  40.0  32.2  −0.33 
(ann) NTs  26.2  20.2  −1.21 
(red) NTs  25.4  18.6  −1.30 

The relevant nature of topography is indicated by Skewness (Sskw): high peaks are implied when Sskw > 0, whereas Sskw < 0 is indicative of valley-like features such as deep scratches [50].

The negative calculated Skewness parameter, Sskw, values (−0.33, −1.21 and −1.30, respectively) show that the samples, either as formed or with the different treatments (annealing, reduction), exhibit low valleys rather than high peaks, reflecting their interspace. Briefly, a negative value for the Skewness parameter translates into the prevalence of valleys in the topography images. The skewness parameter determined for the untreated nanotubes (NTs) was found to be −0.33, suggesting a slight negative asymmetry of the surface. The heights and depths distributions of treated samples show notably greater asymmetry determined by their high Skewness parameters of −1.21 and −1.30, respectively. These results are in agreement with the observed periodic features of the surfaces.

In order to study the adhesive properties of the sample surfaces, a certain number of Force-distance curves were traced using the AFM spectroscopy mode.

This consists of approaching the cantilever to the sample’s surface and as soon as the attractive forces overcome the cantilever spring constant, the tip jumps into contact with the surface. This is the so-called “snap in effect”. The adhesion force was measured from the retracting curve, when the cantilever was pulled away from the surface. The adhesion force variation mappings displayed in Fig. 5c,f,i were determined from the ‘pull off’ region of the force-distance curves (Fig. 4), for more information regarding the measurements and the computation of the adhesion force (F), please see experimental details.

Fig. 4.

Representative examples of the force-distance curves traced on each studied sample: a – as formed NTs; b – annealed NTs; c – reduced NTs; d – TiZr substrate; e – annealed TiZr substrate and f – reduced TiZr substrate.

(0.22MB).
Fig. 5.

2D Topography, Phase and Force Adhesion Variation Mappings, respectively acquired on 1 × 1 μm2 for: a, b, c – as formed NTs, d, e, f – annealed NTs and g, h, i – reduced NTs.

(1.18MB).

In addition, an example of all the steps involved in the AFM analysis, as shown for the reduced nanotubes, is displayed in supporting information Figs. S3–S14. All parameters obtained from the AFM data as well as the contact angles determined for all samples are summarized in Table 3.

Table 3.

Contact Angle (o), Roughness (RMS and Ra) and mean adhesion force values obtained for 1 × 1 μm2 topography scan.

Sample  Contact angle (oRMS (nm)  Ra (nm)  Mean adhesion force value (nN) 
NTs  32.7  34.4  29.0  13.3 
substrate  71  25.8  21.0  31.4 
(ann) NTs  29.6  17.3  13.5  11.2 
(ann) substrate  71  9.7  7.4  30.7 
(red) NTs  78.1  18.4  13.7  9.4 
(red) substrate  71  5.2  4.3  42.1 

Overall, both calcination and reduction, lead to a significant decrease of roughness parameters and adhesion force values of the coatings. The roughness parameter values revealed by the substrates after the thermal treatments suffered a significant decrease, and the adhesive properties showed similar behavior, except for the reduced substrate showing a greater value of the mean adhesion force. To the best of our knowledge, there are no reports published yet in literature on the adhesive properties of highly ordered TiZr-oxide nanotubes after the annealing treatments.

The nanotubular layer significantly improves the reduced elastic modulus and hydrophilicity, as was reported in several papers [51,52], and probably, to some extent it helps to avoid any delamination failure, improving the bone-to-implant contact without the need of an intervening fibrous tissue. For example, the nanotubular surfaces on smooth machined titanium and roughened resorbable blast media treated titanium, significantly reduced water contact angles compared with those prior to anodizing, and the surface roughness parameter value after anodizing increased in the first situation and decreased in the second one [53]. Air annealing of nanotubular structures is a good procedure to decrease the contact angle, but reduction in inert atmosphere decreases the hydrophilic character. Our contact angle determinations and literature data confirm this trend of variations.

Regarding their use in bioapplications, measuring LDH activity (used extensively as an accurate marker for cell death) provides a simple, quick and accurate method for determining cell viability, being an ideal technique for in vitro research on biomaterial biocompatibility [54]. As shown in Fig. 6a, the exposure of gingival fibroblasts to different nanotubes-based coatings on TiZr alloys did not cause significant changes in cell viability even after 72 h of cultivation on the TiZr modified surfaces.

Fig. 6.

Cellular response revealed by the LDH (a) and NO (b) release, and intracellular ROS level (c) after 24, 48 and 72 h cultivation of HGF-1 gingival fibroblasts on nanotubes-based coatings on TiZr alloys. Data are expressed as mean ± standard deviation (n = 3) and showed relative to control (uncoatedTiZr). *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control.

(0.28MB).

Only a small difference could be observed between the uncoated TiZr and annealed NTs. After 24 h of incubation, the level of intracellular lactate dehydrogenase (LDH) released into the culture medium from the cells exposed to the annealed NTs showed a slight increase of only 6% over the control, demonstrating that none of the tested surfaces affected significantly the cell membrane integrity and cell viability in time. To note that recent investigations of reduced TiO2 nanotubes (100 nm diameter) have shown that the reducing treatment did not change the dominant cell attachment response, rat mesenchymal stem cells [45].

The inflammatory potential of these nanotubes coatings grown on TiZr substrates was evaluated using nitric oxide (NO) measurement, and the NO levels released into the culture medium by gingival fibroblasts grown on nanotubular structure on TiZr were very close to the control value, except for the annealed NTs that showed an increase of NO level with almost 10% after 24 h exposure. However, this increase was not significant and overall these results indicated that long-term exposure to nanostructured TiZr alloys did not generate a notable inflammatory response.

The capacity of each biomaterial to induce reactive oxygen species (ROS) is dependent on their particular physical and chemical properties. The generation of ROS and the subsequent production of oxidative stress is a predominant mechanism leading to cytotoxicity and DNA damage [55]. Other studies suggested that ROS are important regulators of cellular signaling processes in fibroblasts, contributing to the formation of a fibrous capsule in response to biomaterial implantation [56].

Therefore, to assess if our TiZr coated samples induced oxidative stress, the level of intracellular ROS was measured using the fluorescence intensity of dichlorofluorescein (DCF). The level of ROS formed in HGF-1 fibroblasts exposed to different treated TiZr surfaces maintained the same pattern after 24, 48 and 72 h. The exposure of gingival fibroblasts to the treated TiZr alloys generated an increase in ROS level by ∼50% for annealed NTs and by ∼20% for NTs. In contrast, annealed NTs determined a slight time-dependent increase of ROS level up to 35% over control. In a previous work, when the TiZr alloy was coated with nanochannels such coatings revealed an improvement of the cell response [57] and recently it was shown to induce a positive effect on osteoblast differentiation and osteoclastogenesis inhibition [58]. These nanochannels were highly hydrophilic (contact angle around 15) and as is well known in majority of cases cells do prefer to adhere to such surfaces, thus improving their biocompatibility [59]. Therefore, we would like to continue our research to examine the influence of calcination and reduction treatments on the adhesive properties of TiZr nanotubes fabricated via anodizing.

Compared with the results on nanochannels, data obtained from the present performed studies suggests as well that the nanotubular anodic coatings on TiZr and with the various post treatment are biocompatible. The experiments with human gingival fibroblast corroborated that after both annealing and reduction, the nanostructures can be used in biomedical applications without generating significant inflammatory processes. According to literature, on nano- and submicron rough titanium and titanium alloys a reduced immune cell response was observed [60]. To note that after the reduction treatment the structure has less hydroxyl groups able to induce hydrophilic character, and the significant increase of the contact angle for reduced NTs simultaneously with maintaining almost the same LDH level after 72 h is an interesting finding indicating a complex interface with almost the same cell behavior based on similar substrate.

Roughness parameter and adhesion force values decreased after the treatments, which might lead to a small increase in the possibility of inflammatory processes appearance at the interface with the tissue. On the other hand, the surface roughness could favor the bacterial adhesion, housing the bacteria within its roughness grooves (having large number of valleys and hills) encountered over the surface [61]. It also should be noted that the decrease of the adhesive properties of the treated surfaces might help in preventing the bacterial adhesion.

4Conclusions

These results are disclosing a new perspective for nanotubular arrays grown on TiZr alloy by electrochemical anodizing, by introducing via the post-preparation treatments the ability of tuning the roughness and adhesive properties in order to make them successfully suitable in different biomedical fields. In this respect, the annealed or reduced nanotubes showed different roughness and hydrophilic balance. Interestingly, regarding the hydrophilic balance of the studied samples, as expected it decreased after annealing but increased after reduction, with only small changes in the cell response. The roughness and wettability can lead to different types of performance, structures that are also suitable for other technological applications.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgments

The work is supported by CNCSIS–UEFISCDI, project PN-III-P4-ID PCE 2016-0316.

Appendix A
Supplementary data

The following are Supplementary data to this article:

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