M. BEN^INA et al.: PERFORMANCE OF ANNEALED TiO2 NANOTUBES IN INTERACTIONS WITH BLOOD PLATELETS 791–795 PERFORMANCE OF ANNEALED TiO 2 NANOTUBES IN INTERACTIONS WITH BLOOD PLATELETS U^INKOVITOST ANATAZNIH TiO 2 NANOCEVK V INTERAKCIJAH S TROMBOCITI Metka Ben~ina 1 , Ita Junkar 1 , Tina Mavri~ 2,3 , Veronika Kralj-Igli~ 3 , Matjaz Valant 4,5 , Ale{ Igli~ 2* 1 Department of Surface Engineering and Optoelectronics, Jo`ef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia 2 Laboratory of Biophysics, Faculty of Electrical Engineering, University of Ljubljana, Tr`a{ka 25, 1000 Ljubljana, Slovenia 3 Laboratory of Clinical Biophysics, Faculty of Health Sciences, University of Ljubljana, Zdravstvena 5, 1000 Ljubljana, Slovenia 4 Materials Research Laboratory, University of Nova Gorica, Vipavska 13, 5000 Nova Gorica, Slovenia 5 Institute of Fundamental and Frontier Sciences, University of Electronic Science and Technology of China, Chengdu 610054, China Prejem rokopisa – received: 2018-11-20; sprejem za objavo – accepted for publication: 2019-05-23 doi:10.17222/mit.2018.249 Titanium dioxide (TiO2) nanotubes, synthesized by the electrochemical anodization of Ti foil, were annealed to obtain the anatase crystal phase and further characterized as surfaces for vascular stent applications. X-ray diffraction (XRD) was used to analyze the crystal structure of the TiO2 nanotubes, while the morphology of the nanotubes and biological material was determined by scanning electron microscopy (SEM). The results showed that the surface of the annealed TiO2 nanotubes acts anti-thrombogenically, since the whole-blood derived platelets do not adhere nor activate readily on such samples, unlike on the surface of amorphous TiO2 nanotubes and plain Ti foil. Therefore, the anatase crystal phase of TiO2 nanotubes could be beneficial for stent applications. Keywords: TiO2 nanotubes, crystallization, blood platelets, vascular stents Predstavljena je sinteza TiO2 nanocevk z metodo elektrokemi~ne anodizacije ter vpliv spremembe kristalne strukture TiO2 nanocevk na interakcije s trombociti. Analiza odziva trombocitov na kardiovaskularnih vsadkih kot so `ilne opornice je klju~nega pomena za njihovo uspe{no integracijo v biolo{ko okolje; oprijem ter aktivacija trombocitov na povr{ini vsadkov sta neza`elena, saj to lahko vodi v nastanek krvnih strdkov. Z rentgensko difrakcijsko spektroskopijo (XRD) smo analizirali kristalno strukturo TiO2 nanocevk, morfologijo pa smo analizirali z vrsti~nim elektronskim mikroskopom (SEM). Rezultati nakazujejo, da se trombociti na povr{ino TiO2 nanocevk z anatazno kristalno strukturo ne oprijemajo, medtem ko je povr{ina amorfnih TiO2 nanocevk ter Ti folije ugodna za oprijem trombocitov. Klju~ne besede: TiO2 nanocevke, kristalizacija, trombociti, `ilne opornice 1 INTRODUCTION Titanium dioxide nanotubes (TiO 2 NTs) manufact- ured via a simple, yet elaborate, anodization process have gained considerable attention for use in photo- catalysis, 1,2 biomedical applications, 3,4 s u c ha sa n accomplished titania platform, biosensing devices, 5,6 implants 7,8 and controlled delivery systems with targeted drug release. 9,10 In the field of implantable materials TiO 2 is at the forefront of real-life applications due to the favourable spontaneous formation of the oxide layer on its surface, which enables high biocompatibility. 11–14 Nevertheless, despite their non-toxic nature, titania- based implants can still be rejected by the host tissue, the response resulting in, e.g., inflammation or thrombosis. 15 Considering TiO 2 NTs, higher biocompatibility is achieved when the anatase or rutile crystal phase prevails in the nanotube arrays. Indeed, it is well known that the crystal structure of the material affects its performance regarding interactions with biological matter. 16 For example, J. A. Sorkin et al. 17 observed the higher proliferation of neuronal stem cells on TiO 2 NTs with a higher percentage of anatase phase. Similarly, increased adhesion and proliferation of osteoblast cells on anatase TiO 2 NTs in comparison to the amorphous counterpart. Other authors observed the same anatase-related trend in osteoblast behaviour. 18,19,20 Anatase-containing TiO 2 NTs with an anatase crystal structure were also reported to promote adhesion and proliferation of human mesen- chymal stem cells. 21 Despite the favourable effects of TiO 2 NTs’ crystallinity on different cells, there are arguments in favour of the hemocompatibility evaluation of blood-contacting devices. Q. Huang et al. 22 showed that rutile-phase TiO 2 NTs reduced platelet adhesion and activation. This particular case is advantageous for vascular stents, for which the inhibition of adhesion and the activation of platelets is preferred, since platelets’ aggregation leads to blood-clot formation and in-stent thrombosis. L. Zhang et al. 23 showed that pure anatase in the TiO 2 NTs, lead to better platelet adhesion, which Materiali in tehnologije / Materials and technology 53 (2019) 6, 791–795 791 UDK 620.3:532.78:549.514.6:616-008.852:616.1 ISSN 1580-2949 Original scientific article/Izvirni znanstveni ~lanek MTAEC9, 53(6)791(2019) *Corresponding author's e-mail: ales.iglic@fe.uni-lj.si (Ale{ Igli~) could be beneficial for the improved integration of dental implants. In the present research, the performance of the annealed TiO 2 NTs in interactions with whole blood- derived platelets is presented. These results could provide an additional basis for explaining the dis- crepancy in the literature concerning platelet adhesion and activation on the annealed TiO 2 NTs. 2 EXPERIMENTAL PART 2.1 Materials Titanium foil (Advent, 0.1 mm thickness, 99.6 %), ethylene glycol (Fluka, 99.5 %), ammonium fluoride – NH 4 F (Sigma Aldrich, 28.0-30.0 %), hydrofluoric acid – HF (Sigma Aldrich, 40 %) acetone (Honeywell Riedel – de Haen, 99.5 %), ethanol (Sigma Aldrich, 96 %), deionized water (miliQ), phosphate-buffered saline – PBS (tablets, Sigma Aldrich), and glutaralde- hyde solution (Sigma Aldrich). 2.2 Synthesis of TiO2 NTs by electrochemical anodi- zation The TiO 2 NTs were fabricated by the electrochemical anodization method, as described in the literature 24,25 with the Voltcraft VSP 2653/VSP 2206 laboratory power supply. Experiments were carried out at room tem- perature ( 20 °C) in a two-electrode system (Pt/Ti foils) with a working distance of 15 mm. Before the anodization process, Ti foil was ultrasonically cleaned in acetone. The Ti foil was then dried under a nitrogen stream. The electrolyte used in the first step of the process was composed of ethylene glycol, NH 4 F (0.35 w/%) and deionized water (1.7 w/%). The nano- tubular layer grown in this step was then removed with a successive ultrasonication in deionized water. In the second step of the anodization process, the pre-treated Ti foil was used as a substrate to grow TiO 2 NTs with a length of 2.53 μm and a tube diameter of 100 nm. The electrolyte based on ethylene glycol, deionized water (11.0 w/%) and HF (1.0 w/%) was used. The morphology of the TiO 2 NTs was controlled by applying a voltage of 58 V. The synthesis time was 2.5 h. The as-synthesized TiO 2 NTs were kept in ethanol for 2 hours in order to remove the electrolyte remains and then dried under a nitrogen stream. The as-prepared TiO 2 NTs were further annealed in a conventional furnace at 450 °C for2hin an air atmosphere with an annealing/cooling rate = 8 °C/min. 2.3 Characterization 2.3.1 Scanning electron microscope (SEM) analysis The morphology of the materials was analysed with a scanning electron microscope (JSM 7100F – JEOL) with an acceleration voltage of 15 keV. The elemental analysis was made with an energy-dispersive X-ray spectrometer (EDX) coupled to a scanning electron microscope. 2.3.2 X-ray diffraction spectroscopy (XRD) analysis The X-ray diffraction (XRD) analysis was performed with a MiniFlex 600 Benchtop X-ray diffractometer (Rigaku) equipped with Cu–K radiation (0.1541 nm) over the 2 range 20–80°, with a step size of 0.017°, divergence slit of 0.218° and counting step time of 25 s in continuous scanning mode. Carbon tape was used to mount the samples on the glass sample holder. 2.4 Interaction of TiO2 NTs with platelets The whole blood was obtained from healthy volun- teers (the authors of the manuscript) via vein puncture. The blood was drawn into 9-mL tubes coated with trisodium citrate anticoagulant (Sigma Aldrich). The number of platelets of whole blood was counted with a multi-parameter automated hematology analyzer (Cell-DYN 3200, Abbott). The material samples – Ti foil, amorphous and annealed TiO 2 NTs (7 mm × 7 mm in size) were incubated with 250 μL of whole blood for 45 min at room temperature ( 20 °C) in the 24-well cell-culture plates. Afterwards, 250 μL of PBS was added to the incubated samples. The blood with PBS was then removed and the samples were rinsed 3 times with 250 μL of PBS in order to remove weakly adherent platelets. The preparation of the samples for SEM analysis was performed in the following manner: the samples were dipped within the solution of 250 μL of PBS and 250 μL of 0.5 vol/% glutaraldehyde solution for 2 h at room temperature. Then the materials were rinsed with PBS and dehydrated by using a graded ethanol series (50, 70, 80, 90, 100) vol/% and again 100 vol/% of ethanol for 5 min and in the last stage (100 vol/% ethanol) for 10 min. Before observation with SEM the samples were dried with liquid nitrogen and left in a vacuum for 3 h. Before the observation with SEM, the samples were coated with gold/palladium. The test was made in triplicates and the representative images are shown. 3 RESULTS The results of the XRD analysis are presented in Figure 1.TiO 2 NTs are amorphous after electrochemical anodization process. The characteristic peaks observed in the XRD pattern of as-anodized TiO 2 NTs belong to the Ti foil, which was used as a substrate for the NTs’ for- mation. The annealing of TiO 2 NTs in the furnace at 450 °C for 2 h induces the transition of the amorphous phase to the anatase crystal structure, as identified from the characteristic peaks (101), (103), (004), (200), (105) and (211). SEM analysis revealed altered morphology of the material annealed TiO 2 NTS which, as evident from Figure 1 and the surface chemistry, as presented in M. BEN^INA et al.: PERFORMANCE OF ANNEALED TiO2 NANOTUBES IN INTERACTIONS WITH BLOOD PLATELETS 792 Materiali in tehnologije / Materials and technology 53 (2019) 6, 791–795 Table 1. The annealed have thicker walls (Figure 2), as already reported elsewhere. 26 X-ray photoelectron spectroscopy (XPS) and energy-dispersive X-ray (EDX) analysis 27 showed the changes in the surface chemistry (Table 1). According to the XPS analysis (depth of analysis of 5 nm) the difference in the concentration of fluorine (F) was observed; annealed TiO 2 NTs do not contain F on the surface, as evident from Table 1, while 8.2 x/% of F is present on the surface of the as-anodized TiO 2 NTs. The fluorine comes from the anodization process and these results indicate that it is only weakly bound or absorbed on the surfaces of the TiO 2 NTs, as after annealing F was not detected on the TiO 2 NTs. A slightly lower concentration of carbon was detected on the as-anodized and annealed Ti foil. Increase in the oxygen concentration compared to the Ti foil was observed for as-anodized NTs (about 43.7 x/%) and as-anodized annealed TiO 2 NTs (about 49.8 x/%). A similar trend was observed for the concentration of Ti, as a slight increase in its concentration was observed on the surface of the annealed TiO 2 NTs. According to results of the EDX (penetration depth 1–2 μm), higher oxygen concentration was detected on the annealed NTs, compared to the as-anodized NTs; about 55 x/% and 64.9 at%, respectively. A slight increase in Ti concen- tration by EDX analysis of the surface of the annealed surface of TiO 2 NTs. F was also detected on the as-anodized NTs, with about 11.9 x/%, while after annealing F was not detected. This confirms that F is indeed distributed along the NTs’ length as newly TiO 2 NTs are about 2.53 μm in length and this corresponds well with the penetration depth of the EDX analysis. XPS analysis showed a similar concentration of C for the as-anodized and annealed TiO 2 NTs, 30.0 x/% and 29.5 x/%, while C was not detected by EDX. This result is in agreement with our previous results of depth profile analysis, which showed that carbon is present only in the top surfacelayer (depth of 2 nm). 27 Interactions between the platelets’ and Ti foil, as anodized and annelaed TiO 2 NTs were studied with SEM, Figure 2. The morphology of the platelets can be categorized as round, dendritic, spread and fully spread. Platelets adhere to the surface of the Ti foil with lamelopodia and filopodia (Figure 2) and their shape can be classified as dendritic. Similar result were observed for the as-anodized NTs. However, platelets could practically not be found on the surface of the annealed NTs. As evident from the SEM image in Figure 2, only one round platelet was observed. This result indicates that the platelets do not adhere to the surface of TiO 2 NTs with an anatase crystal structure, nor does such a surface allow them to activate. 4 DISSCUSSION A sensible approach of TiO 2 NTs crystal structure manipulation to obtain the anatase/rutile phase is by heat treatment in air. Simply by selecting the appropriate annealing temperature, a tailored crystallization and phase composition of either anatase or rutile can be obtained in the TiO 2 NTs. 29 In the present study, annealing of TiO 2 NTs with 100 nm in diameter was performed at 450 °C for 2 h in a conventional furnace. The results showed that such-annealed TiO 2 NTs crystallize into pure crystal structure without significant alteration of NTs morphology. Contrary to a previous report of improved platelet adhesion on the surface of anatase-TiO 2 NTs, 23 the results of the interactions of platelets and TiO 2 NTs anatase phase obtained in the present study show that such surfaces do not allow for platelet adhesion, unlike the surface of amorphous TiO 2 NTs and plain Ti foil (Figure 2). It should be noted the that platelets’ adhesion and activation depend on a variety of other factors, such as morphology, surface chemistry, surface topology, 2 surface charge density and M. BEN^INA et al.: PERFORMANCE OF ANNEALED TiO2 NANOTUBES IN INTERACTIONS WITH BLOOD PLATELETS Materiali in tehnologije / Materials and technology 53 (2019) 6, 791–795 793 Table 1: XPS and EDX analyses of as-anodized and annealed TiO 2 NTs XPS analysis (x/%) COT iNFS i As-anodized NTs* 30.0 43.7 18.2 0 8.2 0 As-anodized – annealed NTs* 29.5 49.8 20.7 0 0 0 EDX analysis (x/%) As-anodized NTs 0 55.0 33.1 0 11.9 0.1 As-anodized – annealed NTs 0 64.9 35.1 0 0 0 * Results taken from reference 28 Figure 1: XRD patterns of as anodized TiO 2 NTs amorphous and annealed TiO 2 NTs; Ti=Ti foil, A=anatase potential, 13,14 wettability, 14 of the material and all these parameters might be altered after annealing. 31 For instance, with increasing of the annealing temperature, the concentration of the cell-toxic electrolyte residual – fluorine decreases. 24,27 In our previous studies, we also proved the long-term hydrophilicity of the annealed TiO 2 NTs, 28 in comparison with the less-hydrophilic amor- phous TiO 2 NTs and hydrophobic Ti foil. 27 Despite the unaltered structure of the TiO 2 NT after annealing, TiO 2 NTs’ surface with anatase crystal structure prepared in the present study do not provide an environment suitable for platelet adhesion and activation, which is from the view point of stent application beneficial, as it could pre- vent platelet aggregation and blood clot formation, i.e., thrombosis. Additional studies about the performance of relevant cells on the surface of TiO 2 NTs are, however, required. Among others, the surface charge density 13 and surface potential 30 of different types of TiO 2 nanotubes should be determined to provide more detailed infor- mation about their interactions with platelets. 5 CONCLUSIONS TiO 2 nanotubes, prepared by electrochemical anodi- zation of a Ti foil, were annealed in order to obtain the transition of the amorphous to anatase crystal phase. The effect of the crystalline material of the interactions with whole-blood-derived platelets was studied. It has been confirmed that annealed TiO 2 NTs with an anatase crystal structure prevent the adhesion and activation of platelets, unlike the amorphous as-anodized TiO 2 NTs and Ti stent for start. From the view point of stent applications, such results are beneficial due to the ability of the materials to prevent thrombosis reactions. Acknowledgment The authors would like to acknowledge the Slovenian Research Agency for financial support, grants No. Z3-4261, J3-9262, J1-9162, J2-8166, J2-8169, J5-7098, P2-0232 and Slovenian Ministry of Education, Science and Sport grant "Public call for encouraging young M. BEN^INA et al.: PERFORMANCE OF ANNEALED TiO2 NANOTUBES IN INTERACTIONS WITH BLOOD PLATELETS 794 Materiali in tehnologije / Materials and technology 53 (2019) 6, 791–795 Figure 2: SEM images of Ti foil, as-anodized TiO 2 NTs and annealed-anatase TiO 2 NTs interacting with platelets investigators at the beginning of their career 2.0", No. 5442-15/2016/18. 6 REFERENCES 1 S. Sreekantan, K. A. Saharudin, L. C. Wei, Formation of TiO2 nano- tubes via anodization and potential applications for photocatalysts, biomedical materials, and photoelectrochemical cell, in: IOP Conference Series: Materials Science and Engineering, IOP Publishing, 2011, 012002, doi:10.1088/1757-899X/21/1/012002 2 L. Qin, Q. Chen, R. Lan, R. Jiang, X. Quan, B. Xu, F. Zhang, Y. Jia, Effect of anodization parameters on morphology and photocatalysis properties of TiO2 nanotube arrays, J. Mater. Sci. Technol., 31 (2015) 1059–1064, doi:10.1016/j.jmst.2015.07.012 3 P. Roy, S. Berger, P. Schmuki, TiO2 nanotubes: synthesis and appli- cations, Angew. Chem. Int. Ed., 50 (2011) 2904–2939, doi:10.1002/ anie.201001374 4 M. Kulkarni, A. Mazare, E. Gongadze, [. Perutkova, V. Kralj-Igli~,I. Milo{ev, P. Schmuki, A. Igli~, M. Mozeti~, Titanium nanostructures for biomedical applications, Nanotechnology, 26, (2015) 062002, doi:10.1088/0957-4484/26/6/062002 5 W.-W. Zhao, Z.-Y. Ma, D.-Y. Yan, J.-J. Xu, H.-Y. Chen, In situ enzymatic ascorbic acid production as electron donor for CdS quantum dots equipped TiO2 nanotubes: a general and efficient approach for new photoelectrochemical immunoassay, Analytical chemistry, 84 (2012) 10518–10521, doi:10.1021/ac3028799 6 G.-C. Fan, L. Han, H. Zhu, J.-R. Zhang, J.-J. Zhu, Ultrasensitive photoelectrochemical immunoassay for matrix metalloproteinase-2 detection based on CdS: Mn/CdTe cosensitized TiO2 nanotubes and signal amplification of SiO2@ Ab2 conjugates, Analytical chemistry, 86 (2014) 12398–12405, doi:10.1021/ac504027d 7 A. Roguska, A. Belcarz, J. Zalewska, M. Holdyn'ski, M. Andrzej- czuk, M. Pisarek, G. Ginalska, Metal TiO2 nanotube layers for the treatment of dental implant infections, ACS Appl. Mater. Interfaces., 10 (2018) 17089–17099, doi:10.1021/acsami.8b04045 8 E. Su, D. Justin, C. Pratt, V. Sarin, V. Nguyen, S. Oh, S. Jin, Effects of titanium nanotubes on the osseointegration, cell differentiation, mineralisation and antibacterial properties of orthopaedic implant surfaces, Bone. Joint. J., 100 (2018) 9–16, doi:10.1302/0301- 620X.100B1.BJJ-2017-0551.R1 9 J. Xu, X. Zhou, Z. Gao, Y.Y. Song, P. Schmuki, Visible-Light- Triggered Drug Release from TiO2 Nanotube Arrays: A Controllable Antibacterial Platform, Angew. Chem. Int. Ed., 55 (2016) 593–597, doi:10.1002/anie.201508710 10 C. Liang, J. Wen, X. Liao, A visible-light-controlled platform for prolonged drug release based on Ag-doped TiO2 nanotubes with a hydrophobic layer, Beilstein. J. Nanotechnol., 9 (2018) 1793–1801, doi:10.3762/bjnano.9.170 11 G. Wang, J. Li, K. Lv, W. Zhang, X. Ding, G. Yang, X. Liu, X. Jiang, Surface thermal oxidation on titanium implants to enhance osteogenic activity and in vivo osseointegration, Sci. Rep., 6 (2016) 31769, doi:10.1038/srep31769 12 L.-H. Li, Y.-M. Kong, H.-W. Kim, Y.-W. Kim, H.-E. Kim, S.-J. Heo, J.-Y. Koak, Improved biological performance of Ti implants due to surface modification by micro-arc oxidation, Biomaterials., 25 (2004) 2867–2875, doi:10.1016/j.biomaterials.2003.09.048 13 E. Gongadze, D. Kabaso, S. Bauer, T. Slivnik, P. Schmuki, U. van Rienen, A. Igli~, Adhesion of osteoblasts to a nanorough titanium implant surface, Int. J. Nanomed., 6 (2011) 1801–1816, doi:10.2147/IJN.S21755 14 M. Lorenzetti, E. Gongadze, M. Kulkarni, I. Junkar, A. Igli~: Elec- trokinetic properties of TiO2 nanotubular surfaces, Nanoscale Res. Lett., 11 (2016) 378, doi:10.1186/s11671-016-1594-3 15 B. S. Smith, S. Yoriya, L. Grissom, C. A. Grimes, K. C. Popat, Hemocompatibility of titania nanotube arrays, J. Biomed. Mater. Res. A., 95 (2010) 350–360, doi:10.1002/jbm.a.32853 16 A. Mazare, M. Dilea, D. Ionita, I. Titorencu, V. Trusca, E. Vasile, Changing bioperformance of TiO2 amorphous nanotubes as an effect of inducing crystallinity, Bioelectrochemistry., 87 (2012) 124–131, doi:10.1016/j.bioelechem.2012.01.002 17 J. A. Sorkin, S. Hughes, P. Soares, K. C. Popat, Titania nanotube arrays as interfaces for neural prostheses, Mater. Sci. Eng. C., 49 (2015) 735–745, doi:10.1016/j.msec.2015.01.077 18 S. Oh, C. Daraio, L.H. Chen, T.R. Pisanic, R.R. Finones, S. Jin, Sig- nificantly accelerated osteoblast cell growth on aligned TiO2 nanotubes, J. Biomed. Mater. Res. A., 78 (2006) 97–103, doi:10.1002/jbm.a.30722 19 Y. Zhang, R. Luo, J. Tan, J. Wang, X. Lu, S. Qu, J. Weng, B. Feng, Osteoblast behaviors on titania nanotube and mesopore layers, Regen. Biomater., 4 (2016) 81–87, doi:10.1093/rb/rbw042 20 A. Roguska, M. Pisarek, A. Belcarz, L. Marcon, M. Holdynski, M. Andrzejczuk, M. Janik-Czachor, Improvement of the bio-functional properties of TiO2 nanotubes, Appl. Surf. Sci. I, 388 (2016) 775–785, doi:10.1016/j.apsusc.2016.03.128 21 K. Khoshroo, T.S. Jafarzadeh kashi, F. Moztarzadeh, H. Eslami, M. Tahriri, The influence of calcination temperature on the structural and biological characteristics of hydrothermally synthesized TiO2 nanotube: in vitro study, Synth. React. Inorg. M., 46 (2016) 1189–1194, doi:10.1080/15533174.2015.1004438 22 Q. Huang, Y. Yang, D. Zheng, R. Song, Y. Zhang, P. Jiang, E.A. Vogler, C. Lin, Effect of construction of TiO2 nanotubes on platelet behaviors: Structure-property relationships, Acta. Biomater., 51 (2017) 505–512, doi:10.1016/j.actbio.2017.01.044 23 L. Zhang, X. Liao, A. Fok, C. Ning, P. Ng, Y. Wang, Effect of crys- talline phase changes in titania (TiO2) nanotube coatings on platelet adhesion and activation, Mater. Sci. Eng. C., 82 (2018) 91–101, doi:10.1016/j.msec.2017.08.024 24 I. Junkar, M. Kulkarni, B. Dra{ler, N. Rugelj, A. Mazare, A. Fla{ker, D. Drobne, P. Humpoli~ek, M. Resnik, P. Schmuki, M. Mozeti~, A. Igli~, Influence of various sterilization procedures on TiO2 nano- tubes used for biomedical devices, Bioelectrochemisty, 109 (2016) 79–86, doi:10.1016/j.bioelechem.2016.02.001 25 I. Junkar, M. Kulkarni, B. Dra{ler, N. Rugelj, N. Recek, D. Drobne, J. Kova~, P. Humpolicek, A. Igli~, M. Mozeti~, Enhanced biocompatibility of TiO2 surfaces by highly reactive plasma, J. Phys. D. Appl. Phys., 49 (2016) 244002, doi:10.1088/0022-3727/49/24/ 244002 26 S. Das, R. Zazpe, J. Prikryl, P. Knotek, M. Krbal, H. Sopha, V. Pod- zemna, J.M. Macak, Influence of annealing temperatures on the properties of low aspect-ratio TiO2 nanotube layers, Electrochim. Acta., 213 (2016) 452–459, doi:10.1016/j.electacta.2016.07.135 27 M. Ben~ina, I. Junkar, T. Lampe, M. Resnik, M. Valant, V. Kralj, M.M. Igli~, A. Igli~, Long-term hydrophilicity of TiO2 nanotubes induced by oxygen plasma treatment, in: VALENTIN^I^, Jo{ko (Ed.). WCMNM 2018 : The Congress incorporates: International Conference on Mult-Materials Micro Manufacturing, (4M), International Conference on Micro Manufacturing, (ICOMM) and Intrenational Forum on Micro Manufaturing, (IFMM). Singapore: Research. 2018, 9–15 28 E. Stojcheva, M. Ben~ina, I. Junkar, T. Lampe, M. Valant, V. Kralj-Igli~, A. Igli~., Visible light responsive TiO2 nanotubes synthesized by electrochemical anodization method. Adv. mat. lett., 9( 2018) 708–714, doi:10.5185/amlett.2018.2024 29 D. Regonini, A. Jaroenworaluck, R. Stevens, C.R. Bowen, Effect of heat treatment on the properties and structure of TiO2 nanotubes: phase composition and chemical composition, Surf. Interface. Anal., 42 (2010) 139–144, doi:10.1002/sia.3183 30 S. Mohajernia, A. Mazare, E. Gongadze, V. Kralj-Igli~, A. Igli~, P. Schmuki, Self-organized, free-standing TiO2 nanotube membranes: Effect of surface electrokinetic properties on flow-through mem- branes, Electrochim. Acta., 245 (2017) 25–31, doi:10.1016/ j.electacta.2017.05.115 M. BEN^INA et al.: PERFORMANCE OF ANNEALED TiO2 NANOTUBES IN INTERACTIONS WITH BLOOD PLATELETS Materiali in tehnologije / Materials and technology 53 (2019) 6, 791–795 795