Electrophoretic deposition of nanocrystalline TiO2 particles on porous TiO2-x ceramic scaffolds for biomedical applications

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Electrophoretic deposition of nanocrystalline TiO2 particles on porous TiO2-x ceramic scaffolds for biomedical applications Inga Narkevica a,∗ , Laura Stradina a , Liga Stipniece a , Eriks Jakobsons b , Jurijs Ozolins a a Rudolfs Cimdins Riga Biomaterials Innovations and Development Centre of RTU, Institute of General Chemical Engineering, Faculty of Materials Science and Applied Chemistry, Riga Technical University, Pulka St. 3, Riga LV-1007, Latvia b Pauls Stradins Clinical University Hospital, Cell Transplantation Centre, Pilsonu St. 13, Riga LV-1002, Latvia

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Article history: Received 26 January 2017 Received in revised form 21 March 2017 Accepted 24 March 2017 Available online xxx Keywords: Titanium dioxide Bioceramic Scaffold Electrophoretic deposition

a b s t r a c t Nanocrystalline TiO2 coated scaffolds offers the possibility to be used in bone tissue regeneration providing not only space for new tissue formation, but also to enhance bioactivity of the implant. In the present study, direct current electrophoretic deposition (EPD) was chosen as simple and low cost technique to coat 3D porous structure of TiO2-x ceramic. Suspension for EPD was prepared suspending nanocrystalline TiO2 particles in isopropanol and adding triethanolamine as dispersant. TiO2 particles were electrophoretically deposited on the surface of TiO2-x scaffolds through varying EPD time and applied voltage. The scaffold pore structure was maintained after applying the coating by EPD. The deposition of nanocrystalline TiO2 coating can be a smart strategy to impart bioactive properties to the 3D scaffold, allowing formation of spherical hydroxyapatite particles on the coated scaffolds after immersion in simulated body fluid. In vitro cell studies does not show cytotoxic effect of nanocrystalline TiO2 coated scaffolds. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction Titanium dioxide (TiO2 ) is present in nature mainly in three crystalline modifications: rutile, anatase and brookite [1]. Thanks to its unique properties, TiO2 is widely used in many technological fields: water purification [2], sensors [3], photocatalyst [4], photovoltaics [5], corrosion protective coatings [6], biomaterials [7–9], antimicrobial coatings [10,11], etc. Special interest is given to nanostructured TiO2 in anatase phase because it have the highest performance in terms of catalytic, photocatalytic activity and gas-sensing properties [12,13]. Biomedical applications of TiO2 photocatalysis are also of increasing interest primarily with respect to antibacterial sterilization and antifungal and antiviral effects [14]. As photocatalysis is an interfacial phenomenon, maximized surface-area-to-volume ratio of TiO2 will optimize it [11]. Apart from photocatalytic properties, another unique feature of nanosized TiO2 is superhydrophilicity, induced by UV irradiation [15]. These all features are dependent from TiO2 particle size, thus many techniques are developed for nanosized TiO2 coating production, such as sol-gel, hydro-thermal, solvo-thermal and chemical vapor deposition [16]. Among other technologies used to produce nanostructured TiO2 coatings, the electrophoretic deposition (EPD)

∗ Corresponding author. E-mail address: [email protected] (I. Narkevica).

technique has emerged as one of the most promising technologies due to its versatility, simplicity and low cost [17]. EPD is based on the motion of charged particles in suspension under the influence of an electric field towards an oppositely charged electrode [17,18]. Changing EPD parameters (time and voltage) it is easy to adjust thickness and morphology of the coating [18]. EPD can be used to obtain coatings on conductive substrates (metals, carbon/graphite, ceramic materials) or on substrates near the electrode surface [18,19]. In the cases, when the substrate to be coated is not electrically conductive, strictly speaking EPD is not occurring, thus term electrophoretic impregnation is applied [20]. One of the major advantage of EPD is the possibility to coat substrates with different structure and shape [21]. The deposition on threedimensional and conducting porous materials have been reported for example by Noberi et al. [22] and Zaman et al. [23] using filters as substrates for TiO2 coating. Due to the TiO2 biocompatibility with the human body, TiO2 based biomaterials are largely employed as bone substitutes, particularly as scaffolds for bone tissue regeneration [7,24]. 3D porous scaffolds can provide a sustainable microenvironment for new bone formation and in the same time long term mechanical support in case of large bone defects, while bioresorbable implant material could be resorbed by the body before the completion of osteogenesis [25,26]. To obtain mechanically resistant TiO2 ceramic scaffolds, they must be sintered at high temperatures that leads to grain growth [27,28]. But as mentioned in literature, nanoscale TiO2

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Please cite this article in press as: I. Narkevica, et al., Electrophoretic deposition of nanocrystalline TiO2 particles on porous TiO2-x ceramic scaffolds for biomedical applications, J Eur Ceram Soc (2017), http://dx.doi.org/10.1016/j.jeurceramsoc.2017.03.053

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enhance implant integration with host tissue in orthopaedic applications [29]. By applying TiO2 nanoparticle coating on porous TiO2 scaffolds it is possible to improve also in vitro bioactivity or bonelike apatite forming ability in simulated body fluid [30,31]. Literature survey points out the necessity of nanosized TiO2 due to enhanced properties not only in biomedical application, but also in other technological fields. For example, nanosized TiO2 coated foams can be used in water disinfection, as the contact efficiency of the bacterial cell and photocatalyst can be increased due to the lightweight and 3D porous structure of the foams and nanosized TiO2 coating [32]. Taking into account the aforementioned statements, we believe that nanosized TiO2 coated foams offers the possibility to be employed not only to enhance bioactivity of the implant, but also as a photosensitive coating to prevent bacterial infections before and during the implantation, as well as for photocatalytic applications. To the best of the authors’ knowledge, no attempt at deposition TiO2 nanoparticles on 3D highly porous TiO2 substrates by EPD has not been reported. Thus, the goal of present work was to investigate the influence of EPD parameters on the properties of obtained nanocrystalline TiO2 coatings on porous TiO2-x ceramic electrodes and assess in vitro bioacitivity and cytocompatibility of produced scaffolds. 2. Materials and methods 2.1. TiO2 scaffold production A flowchart of the scaffolds production is given in Fig. 1. Detailed information about electrically active TiO2-x ceramic scaffolds can be found in [33]. Porous TiO2 scaffolds were produced via polymer foam replica method using cylindrical PU foams (80 ppi, Vitabaltic International) with fully interconnected pore structure as a sacrificial template. Commercially available TiO2 anatase powder (puriss., Sachtleben Chemie GmbH, HOMBITAN LW – S) with average particle size of 180 nm (65 wt.%), 5 wt.% polyvinyl alcohol solution (PVA, 88% hydrolyzed, Mw = ∼25,000, Polysience) (4 wt.%), ethylene glycol (puriss. p.a., Sigma-Aldrich) (6 wt.%), and deionised H2 O (25 wt.%) were used as raw materials to obtain ceramic slurry. Homogenization of ceramic slurry was conducted by vigorous stirring at 1000 rpm for 3 h, and pH of the slurry was kept above 10 using 25% ammonia solution. Further, PU sponges were immersed in prepared slurry and compressed slightly to facilitate its absorption, excessive slurry was removed by squeezing. After drying at ambient temperature for 24 h, coated PU foam templates were thermally treated in air in an electric furnace using a 2-stage schedule, which included heating from 25 ◦ C to 450 ◦ C at a heating rate of 0.5 ◦ C/min, dwelling at 450 ◦ C for 1 h, further heating from 450 ◦ C to 1100 ◦ C at 3 ◦ C/min, and dwelling at 1100 ◦ C for 10 h and finally cooling down to 25 ◦ C. To obtain mechanically resistant ceramic scaffolds, additional sintering at 1500 ◦ C for 30 h was provided. In order to obtain electroconductive TiO2-x scaffolds additional thermal treatment under high vacuum conditions (10−4 Pa) at 1200 ◦ C for 5 h was applied. 2.2. Electrophoretic deposition of nanocrystalline TiO2 Nanocrystalline TiO2 powder in anatase phase (Nanostructured & Amorphous Materials Inc.) with average particle size of 15 nm was used for EPD. The suspension was prepared by adding 0.2 g of TiO2 powder to 100 mL isopropanol (Sigma-Aldrich) which contains triethanolamine (concentration from 0 to 40 mL/L, TEA, Sigma-Aldrich) as a dispersant and magnetically stirred for 1 h followed by ultrasonification for 5 min (24 kHz). Electroconductive TiO2-x scaffold (size ∼3.3 × 40 mm) and titanium plates were used as electrodes for direct current EPD processes (Fig. 2). Electrodes

were cleaned with ethanol in an ultrasonic bath for 5 min and dried. The distance between electrodes was set to 1 cm. EPD experiments were carried out varying deposition time (5–30 min) and applied voltage (5–30 V) in order to determine optimal EPD parameters for high quality of TiO2 coating on the scaffolds. The quality of the coatings was assessed by SEM and it was related to the homogeneity of the coating microstructure and to the extent of uniform covering of the scaffold surface. The coated scaffolds were thermally treated in air at 700 ◦ C for 1 h.

2.3. Material characterization Dilatometric properties of nanocrystalline TiO2 powder in the range of room temperature to 1350 ◦ C were determined with a heating microscope “Hesse Instruments 0–1650 ◦ C” (heating rate 15 ◦ C/min). The phase composition of the scaffolds was evaluated using X-ray diffractometer (XRD, PANalytical X’Pert PRO). Cu K␣ filtered radiation in 2␪ range from 20◦ to 70◦ was used. The microstructure of the samples was examined by the field emission scanning electron microscopy (FE-SEM, Mira/LMU, Tescan) at an acceleration voltage of 15 kV and a distance of 10 mm. Samples were sputter coated with a thin gold layer (thickness 15 nm). Fourier transform infrared spectroscopy (FTIR, Varian 800 FT-IR) was used to analyse the functional groups on the samples before and after immersion in simulated body fluid (SBF), by recording absorbance spectra applying KBr pellet method.

2.4. In vitro bioactivity test and cell studies Immersion in SBF and evaluation of apatite like layer formation on the surface is often used to assess the bioactivity of biomaterials [8]. SBF was prepared by dissolving reagent-grade NaCl, NaHCO3 , KCl, K2 HPO4 ·3H2 O, MgCl2 ·6H2 O, CaCl2 , Na2 SO4 (Sigma Aldrich) in distilled water and pH 7.40 ± 0.2 was set with tris(hydroxymethyl)-aminomethane ((CH2 OH)3 CNH2 ; Sigma Aldrich) and 1 M HCl (Sigma Aldrich) at 36.5 ± 0.5 ◦ C. Each sample was immersed in 100 mL of SBF at 36.5 ± 0.5 ◦ C and SBF was refreshed every other day. After immersion in SBF for 21 days, samples were removed from the SBF, gently washed with deionised water, dried at room temperature and stored in desiccator until further characterization. In vitro cell studies were conducted using MG-63-GFP cell line human osteoblast that was obtained from American Type Culture ® Collection (ATCC CRL-1427TM , Rockville, MD, USA) and maintained in DMEM containing 10% FBS and 2 mM L-glutamine (all from Thermo Fisher Scientific). Cell line was incubated in thermostat at 37 ◦ C with 5% CO2 in a humified atmosphere. Before cell seeding sample conditioning was performed by dipping scaffolds (24 h) in growth media to promote removal of air trapped bubbles. Each sample was seeded with ∼ 200,000 MG-63-GFP cells and incubated for 72 h at 37 ◦ C and 5% CO2 . As a control 200 000 cells were incubated in the wells without samples for 72 h at 37 ◦ C and 5% CO2 . After 3 days of proliferation cells were harvested and cytotoxicity ® was analysed using DNA quantification assay CyQUANT (Thermo Fisher Scientific, USA). Cell proliferation dynamics was monitored by florescent microscopy. Samples after cell seeding and incubation were fixed using 4% PFA (Sigma Aldrich, USA) and dehydrated by a graded ethanol series from 10 to 100%, with three times 20 min incubation at each step for SEM analysis. Samples were sputter coated with gold layer before microscopic investigations.

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Fig. 1. Flowchart of scaffold production.

3. Results and disscusion 3.1. Electrophoretic deposition and characterization of produced scaffolds The typical macrostructure of the produced TiO2 and TiO2-x scaffolds are shown in Fig. 1. The initial pore network of the PU foam templates was well replicated without blocking of the pores. TiO2-x ceramic scaffolds can be characterized with fully open and interconnected pore structure. The pore size was in the range from 70 to 350 ␮m. Porosity of the TiO2 and TiO2-x scaffolds was around 95%, as calculated by geometric method. Compressive strength of the scaffolds was 0.4 ± 0.1 MPa. The macrostructure of TiO2 scaffolds was not affected by thermal treatment under high vacuum conditions

due to the much lower temperature (1200 ◦ C comparing to sintering temperature in air (1500 ◦ C)). As described in [33], thermal treatment under high vacuum conditions greatly increases electrical conductivity of the scaffolds from ∼10−9 mS/m to ∼40 mS/m that is comparable to semiconductors and thus allowing to use TiO2-x ceramic scaffolds in EPD as substrate/electrode. 3D porous TiO2-x scaffolds were coated with nanocrystalline TiO2 particles using electrophoretic deposition. Commercially available nanocrystalline TiO2 powder with average particle size of 15 nm and specific surface area ∼180 g/m2 was used for EPD. Nanopowder was received in dry form and as evident from SEM micrograph (Fig. 3) powder contained agglomerates in few micron size. Ultrasonification was used to break TiO2 agglomerates in suspension and different amount of TEA was added to stabilize the

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Fig. 2. Experimental setup for electrophoretic deposition on porous TiO2-x scaffolds.

Fig. 3. SEM micrograph of nanocrystalline TiO2 powder.

particles. TEA stabilize TiO2 nanoparticles in isopropanol based on electrostatic stabilization mechanism, but its aliphatic chains may also contribute to some steric stabilization mechanism [34]. SEM micrographs (Fig. 4) showed that after sonification and without TEA addition suspension was very unstable and settling of particles after few hours was observed, indicating reagglomeration of the particles. Most stable suspension with smallest particle size was obtained using TEA 10 mL/L, but still some agglomerated particles were observed. For further EPD experiments suspension with 10 mL/L TEA was used.

Realizing electrophoretic deposition on porous TiO2-x electrodes, TiO2 particles were deposited on the surface of ceramic walls, and open and interconnected pore structure of the scaffolds was maintained (Fig. 2As shown in Fig. 5), deposition yield increased linearly by increasing deposition time or applied voltage. If the electrophoretic deposition time was too short (below 10 min), the surface of ceramic walls was not completely covered with TiO2 nanoparticles (Fig. 6). Increasing deposition time relatively uniform coating can be obtained. Also by using too low voltage (below 10 V) ceramic walls were not completely covered (Fig. 7). If the voltage is to high (30 V), TiO2 particles deposit on the nearest surface that are on their migration way forming thick layer on the one side of the wall and the other side leaving uncoated (Fig. 7). Satisfactory results were obtained and the inner and outer parts of the porous TiO2-x scaffolds were homogeneously covered by nanocrystalline TiO2 particles at 20 V for 20 min. An increase in the deposition time to 30 min at 20 V induced rapid TiO2 particle deposition at the edges of the electrode and filling the open pores of the substrate as shown in Fig. 8. Using shorter deposition time such phenomena was not observed. The non-uniform coating during increased deposition time might have been caused by increased deposition resistance and decreased suspension concentration in the inner parts of the scaffold. The already formed coating can act as an insulator layer, affecting the deposition. The other reason could be that after immersion in the suspension and during EPD, the particles that were in-between the pores deposited on the surface and further particle migration in the depth of the scaffold was slower leading to denser coting on the outer layers. At short deposition time and low applied voltage cracking of the deposited TiO2 coating was not observed. But as evident from Fig. 8, if the coating was thicker some cracks are observed. Cracking may

Fig. 4. SEM micrographs of nanocrystalline TiO2 suspensions with different amount of TEA.

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Fig. 5. Deposition yield of the nanosized TiO2 coating against (A) deposition time (V = 20 V) and (B) deposition voltage (t = 20 min) on porous TiO2-x electrodes.

Fig. 6. SEM micrographs of EPD coated scaffolds varying deposition time.

appear due to the evaporation of the used solvent from the coating surface during the drying process. The optimum parameters for used EPD system in order to coat TiO2-x scaffolds are considered 20 V for 20 min. Thermal treatment of the TiO2 coating was performed to improve coating adhesion to the substrate and to eliminate TEA from the coating that could have undesirable impact on further application of the scaffolds. As evident from TiO2 nanopowder dilatometric curve shown in Fig. 9B, sintering of the powder began at ∼550 ◦ C, thus lower temperatures should not be used. XRD spectra shows that during the sintering changes crystalline modification of the powder. Deposited TiO2 particles are in anatase phase, but during sintering phase transformation from anatase to rutile crystallographic modification occurs (Fig. 9B). Increasing sintering temperature up to 1000 ◦ C temperature, pure rutile phase is formed. By sintering coated scaffolds at 700 ◦ C anatase phase of the coating can be remained. From SEM micrograph of the coated TiO2 scaffold surface after thermal treatment at 700 ◦ C (Fig. 10) showed that thermal treatment does no induce significant cracking and grain growth of the coating, grains are still in the nanoscale.

3.2. In vitro bioactivity assessment in SBF Earlier TiO2 has been considered as bioinert ceramic [35], but recent studies have shown that it is possibly to obtain bioactive TiO2 [36–39]. Bioactive materials in the living body form biologically active hydroxyapatite layer on the implant surface and bond to living bone through the apatite layer [40]. The bioactivity of TiO2 scaffolds have been improved by coating it with different ® bioactive polymer/inorganic material (hydroxyapatite, Bioglass ) composites [41,42]. Our previous studies showed that by coating TiO2 scaffolds with TiO2 nanoparticles using vacuum impregnation method in vitro bioactive samples can be produced [30]. The reason is that TiO2 bioactivity strongly depend on the TiO2 particle size [43,36]. Previous researches [30,36,43] have shown that the nanostructured grains have higher surface energy, higher surface area due to smaller particle size and more Ti-OH groups on the surface and thus it can induce apatite deposition. In vitro assessment of bioactivity was carried out on the TiO2 scaffolds and after coating with TiO2 nanoparticles by EPD (EPD parameters: 20 V, 20 min) and sintering at 700 ◦ C temperature. Sintering temperature (700 ◦ C) was chosen based on the previous

Please cite this article in press as: I. Narkevica, et al., Electrophoretic deposition of nanocrystalline TiO2 particles on porous TiO2-x ceramic scaffolds for biomedical applications, J Eur Ceram Soc (2017), http://dx.doi.org/10.1016/j.jeurceramsoc.2017.03.053

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Fig. 7. SEM micrographs of EPD coated scaffolds varying applied voltage.

Fig. 8. SEM micrographs of middle and edge of EPD coated TiO2-x scaffold at 30 min and 20 V.

Fig. 9. (A) Dilatometric curve of TiO2 nanopowder and (B) XRD patterns of commercial TiO2 nanopowder: (a) as received, (b) sintered at 700 ◦ C, (c) 1000 ◦ C,)- and (d) TiO2 scaffold.

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Fig. 10. SEM micrographs of nano TiO2 coated scaffolds (EPD parameters: 20 V, 20 min) after thermal treatment at 700 ◦ C.

Fig. 11. (A) SEM micrographs and (B) FT-IR spectra of the TiO2 and nano TiO2 coated scaffolds after immersion in SBF for 21 days.

research [43], where the impact of nanocrystalline TiO2 powder sintering temperature in the range from 600 ◦ C to 900 ◦ C on apatite-forming ability was evaluated. Results indicated that lower sintering temperature enhances apatite formation on the TiO2 ceramic surface. However, in the current research sintering temperature of 700 ◦ C was chosen due to the fact that at higher temperature sintering of TiO2 ceramic intensifies, but it still possesses in vitro bioactivity. SEM observation (Fig. 11A) revealed that TiO2 scaffolds did not induce apatite deposition on the surface after 21-day soaking in SBF, while the formation of spherical particles on the surface of nano TiO2 coated scaffolds was clearly observed. The ceramic walls of coated scaffolds were completely covered with newly formed particles. Fig. 11B shows FT-IR spectra of the scaffolds after immersion in SBF for 21 days. A broad absorbance band in the range from 3300 to 3700 cm−1 indicate the presence of adsorbed water. Absorbance bands between 950 and 1100 cm−1 attributed to phosphate groups [PO4 ] were observed on coated scaffolds, indicating the presence of apatite structure. A broad ␯3 [CO3 ] absorption band in the region

1350–1570 cm−1 confirmed that deposited apatite was partly carbonated. ␯2 [CO3 ] absorption band at 872 cm−1 indicated formation of B type CO3 2− substituted apatite. The FT-IR absorption bands from 500 to 700 cm−1 were not used for characterization due to overlapping of characteristic absorption bands of TiO2 and apatite. FT-IR spectra revealed that the sphere like particles deposited on the coated scaffolds were carbonate-substituted hydroxyapatite or bonelike apatite. It can be concluded from above results that the nano TiO2 coated scaffolds have apatite-inducing ability. 3.3. In vitro cell studies Impact of produced scaffolds on cell behaviour was characterized via cell morphology and proliferation. The responses to TiO2 scaffold, TiO2-x scaffold and nano TiO2 coated scaffold were compared. Fig. 12 shows fluorescence microscopy images of osteoblasts MG-63-GFP on the scaffold surface after 3-day incubation period. Osteoblasts are spread throughout entire structure of the scaffolds and had an elongated cell shape forming many cellular extensions.

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Fig. 12. Fluorescence images of osteoblast MG-63-GFP cells on TiO2 , TiO2-x and nano TiO2 coated scaffold surfaces after 3-day incubation in culture medium.

scaffolds is similar to TiO2 or TiO2-x scaffolds. In summary, these results pointed out a cytocompatibility of the produced scaffolds. 4. Conclusions Highly porous TiO2 scaffolds with fully open and interconnected pore structure were prepared using polymer replica method. After thermal treatment of the TiO2 scaffolds under high vacuum condition electrically active TiO2-x ceramic was produced that allowed to use it as electrode/substrate in electrophoretic deposition. TiO2-x ceramic scaffolds were successfully coated with nanocrystalline TiO2 particles by the electrophoretic deposition technique. Optimizing deposition parameters relatively smooth coating that is uniformly distributed throughout the scaffold walls and struts without blocking the macropores of the scaffolds was obtained. The best quality of nanocrystalline TiO2 coating on the scaffolds was obtained at 20 V for 20 min. Thermal treatment of the coating induced phase transformation from anatase to rutile crystalline modification, thus as optimal treatment temperature was chosen 700 ◦ C. The in vitro bioactivity of the nano TiO2 coated scaffolds was assessed by the formation of spherical particles of hydroxyapatite nanocrystals after soaking in SBF for 21 days, which were not observed on uncoated scaffolds. In vitro cell tests indicated the cytocompatibility of the produced scaffolds. Acknowledgements Support for this work was provided by the Riga Technical University through the National Research Programme “Multifunctional Materials and composites, photonics and nanotechnology (IMIS2 )” Project No. 4 “Nanomaterials and nanotechnologies for medical applications”. Fig. 13. (A) Proliferation of osteoblast MG-63-GFP cells on TiO2 , TiO2-x and nano TiO2 coated scaffolds after 3-day incubation in culture medium (***p < 0.001, *p > 0.05) and (B) SEM micrograph of osteoblast morphology on nano TiO2 coated scaffold surface.

Formation of the cell clusters in between the ceramic walls was observed. DNA quantification was used to determine the number of cells growing on the produced scaffolds after 3 days of culture (Fig. 13A). As evident after 3 days of culture the number of cells was slightly lower compared to control. No statistical significance was observed between the produced scaffolds. The number of cells on the scaffolds after 3-day culture increased almost two times. As evident from SEM micrograph (Fig. 13B) osteoblasts on nano TiO2 coated scaffolds presented different cellular shape – from rounded to elongated cell shape with many protrusions or filopodia connecting to the other cells. Some TiO2 nanoparticle uptake by osteoblast cells on nano TiO2 coated scaffolds can be seen that requires further studies. Nevertheless, cell viability on the nano TiO2 coated

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Please cite this article in press as: I. Narkevica, et al., Electrophoretic deposition of nanocrystalline TiO2 particles on porous TiO2-x ceramic scaffolds for biomedical applications, J Eur Ceram Soc (2017), http://dx.doi.org/10.1016/j.jeurceramsoc.2017.03.053