Biocompatibility and Surface Properties of TiO2 Thin Films Deposited by DC Magnetron Sputtering

We present the study of the biocompatibility and surface properties of titanium dioxide (TiO2) thin films deposited by direct current magnetron sputtering. These films are deposited on a quartz substrate at room temperature and annealed with different temperatures (100, 300, 500, 800 and 1100 °C). The biocompatibility of the TiO2 thin films is analyzed using primary cultures of dorsal root ganglion (DRG) of Wistar rats, whose neurons are incubated on the TiO2 thin films and on a control substrate during 18 to 24 h. These neurons are activated by electrical stimuli and its ionic currents and action potential activity recorded. Through X-ray diffraction (XRD), the surface of TiO2 thin films showed a good quality, homogeneity and roughness. The XRD results showed the anatase to rutile phase transition in TiO2 thin films at temperatures between 500 and 1100 °C. This phase had a grain size from 15 to 38 nm, which allowed a suitable structural and crystal phase stability of the TiO2 thin films for low and high temperature. The biocompatibility experiments of these films indicated that they were appropriated for culture of living neurons which displayed normal electrical behavior.

has less variability than the in vivo tests and facilitates the experimental replication. In order to use TiO 2 thin films in the biomedical microsensors fabrication, these films must satisfy the in vitro biocompatibility tests. Next, these microsensors must be satisfactory evaluated in vivo animals. Finally, they could be used in clinical studies. Thus, we present a biocompatibility analysis and surface characterization of TiO 2 thin films deposited on a quartz substrate, which are generated by reactive DC magnetron sputtering and annealing at different temperatures.

Physical Properties of the TiO 2 Films
The TiO 2 thin films are characterized using an X-ray diffractometer (XRD) at room temperature. The microstructural properties of the films were measurement using XRD (Bruker, D8 Advance, Karlsruhe, Germany) with CuKα radiation in the 2θ degree angle between 20° and 80°. Figure 1 shows the XRD data of TiO 2 thin films annealed to temperatures between 100 and 1100 °C. The X-ray diffraction pattern of the TiO 2 thin films shows a maximum signal about 20°, which is due to the quartz substrate. Similar X-ray diffraction patterns are obtained of TiO 2 thin films annealed between 100 and 300 °C. However, X-ray diffraction patterns of TiO 2 thin films annealed at 500 °C showed sharp diffraction peaks that can be assigned to the anatase TiO 2 phase with tetragonal body-centered structure (ICDD: 00-021-1272) [38]. In addition, the X-ray diffraction pattern of the TiO 2 thin film annealed at 800 °C showed the coexistence of anatase-rutile, in where the intensity of rutile phase over anatase phase increased due to increment of the thermal annealing treatment. The rutile TiO 2 phase (ICDD: 01-089-4920) [39] showed a tetragonal body-centered structure and a crystallographic orientation in plane (110). Finally, TiO 2 thin film annealed at 1100 °C had a set of sharp diffraction lines of the rutile TiO 2 phase. We conclude that the films have a strong orientation in the direction normal to the substrate surface.
The average crystalline grain size of anatase and rutile phase of TiO 2 thin films are shown in Table 1. The increment of the thermal annealing treatment caused that the grain size of the anatase and rutile TiO 2 phase had an increased and decreased, respectively. The crystalline grain size is obtained by the X-ray line profile analysis through of the Warren-Averbach method using a Pearson VII function (McKeehan) [40].

Biocompatibility Test
The control glass substrate and TiO 2 thin film surfaces of the neurons displayed typical membrane ionic currents activated by voltage variations, including a fast inward current at the beginning of voltage clamp pulse (Na + current) and ensuing outward currents (K + currents). This set of membrane ionic currents constitute the basis of the electrical excitability of neurons. The Na + current form the upstroke of the action potential and the ensuing K + currents repolarize the membrane (Figure 2A,C,E). The inward and outward current density of the dorsal root ganglion (DRG) neurons of Wistar rats cultured in the control substrate and TiO 2 thin films are evaluated. The current density is obtained dividing the current amplitude with respect to the membrane capacitance. The inward and outward current are measured at −20 and +40 mV, respectively.
The inward current density is significantly less for DRG neurons grown on the TiO 2 thin film surfaces prepared at room temperature and at 300 and 1100 °C than that for DRG neurons grown on the control substrate (Table 2 and Figure 3A). Regarding other TiO 2 thin films annealed at temperatures of 100, 500 and 800 °C, there is no significant difference compared with control substrate ( Table 2 and Figure 3A). The amplitude of the outward current density of the DGR neurons cultured in the TiO 2 thin films surfaces at room temperature and 1100 °C are lower than those obtained for neurons cultured on the control substrate (Table 2 and Figure 3B). The outward current density in other TiO 2 thin films is not significantly different with respect those measured of the control substrate (Table 2 and Figure 3B).
In current clamp experiments, the membrane voltage of the cells is about −60 mV and under depolarizing current pulse injection, the DRG neurons are capable of firing typical action potentials of similar morphology both in control substrate and TiO 2 films surfaces ( Figure 2B,D,F).
To analyze the properties of action potentials, we recorded the response to current pulses of DRG neurons grown on the control substrate and TiO 2 thin films surfaces ( Figure 4). The most of the action potential parameters measured of the TiO 2 films surfaces are not significantly different from those measured in the control substrate (Table 3). Only the action potential depolarization rate measured of the TiO 2 film surface annealed at 500 °C (n = 6) is significantly lower than that of the control substrate (Table 3; p = 0.039). In addition, the threshold of the TiO 2 films surfaces annealed at 100 and 500 °C are significantly lower than that of the control substrate (n = 5, p = 0.013 and n = 6, p = 0.017 respectively).
In the control glass substrate and TiO 2 films annealed at room temperature and 100 °C, the 37% of the DRG neurons produced repetitive firing when were subjected to depolarizing current pulse injection. In the other TiO 2 films only 33% of the DRG neurons produced two or more action potential.

TiO 2 Films Deposition
TiO 2 thin films are deposited on a quartz substrate at room temperature by DC magnetron sputtering using a titanium target, which has a diameter of 50.8 mm. A TiO 2 ceramic material is located on a 20% of the titanium target surface, in which both materials registered a purity of 99.99%, respectively. First, the quartz substrate is cleaned using ultrasonic baths of C 3 H 6 O, C 2 H 6 O, and distilled water during 5 min at room temperature. This procedure is repeated four times and, after, the TiO 2 thin films depositions are made under an Argon (Ar) atmosphere and a chamber pressure of 5.6 × 10 −6 Torr. The Ar flow is kept to 15 standard cubic centimeters per minute (sccm) during the TiO 2 films deposition by DC magnetron sputtering. The DC power and substrate temperature are controlled to 100 W and 25 °C, respectively. Figure 5 shows a reactive DC magnetron-sputtering system, which is used to deposit TiO 2 thin films on a quartz substrate (25.4 mm × 25.4 mm). Next, TiO 2 films are subjected to thermal-annealing treatment to achieve their phase transformation. For this treatment, a thermo scientific thermolyne muffle furnace (model F48025-60-80, Thermo Fisher Scientific Inc., Waltham, MA, USA) is used to keep the temperature during one hour in each heat treatment. The time of each heat treatment is lower than that reported in elsewhere [40]. In addition, it is enough to reach the required recrystallization and transformation phases. Finally, TiO 2 thin films are annealed at different temperatures (100, 300, 500, 800 and 1100 °C) to the anatase-to-rutile phase transformation.

Electrophysiological Recording and Data Analysis
Neurons of the dorsal root ganglia are identified under a phase contrast microscope (Nikon TMS, Tokyo, Japan) and classical patch clamp methods are used for electrophysiological recordings [42]. Patch clamp pipettes are pulled from borosilicate glass capillaries using a Flaming-Brown (80/PC; Sutter Instruments, San Rafael, CA, USA). Commonly, these pipettes had a resistance of 1 to 3.5 MΩ and the seal resistance to cell membrane exceeded 1 GΩ. We used a voltage clamp amplifier Axopatch 200B (Molecular Devices, Sunnyvale, CA, USA). Command signals and data acquisition are generated by an analog-digital converter (Digidata 1200, Molecular Devices), which is controlled by the pClamp 10 software (Molecular Devices) [43]. Both voltage and current clamp experiments are performed. In these experiments, cell capacitance and series resistance are electronically compensated (80%) when the whole-cell configuration is obtained. Electrophysiological signals are low-pass-filtered at 5 or 2 kHz and digitized at 20 or 10 kHz. Cells are bathed with standard external and internal solutions, as shown in Table 4. In addition, internal solutions contain both 2 mM ATP-Mg and 1 mM GTP-Na. The pH levels of the external and internal solutions are adjusted at 7.4 with NaOH and 7.2 with KOH, respectively. Osmolarity is monitored by a vapor pressure osmometer (Wescor, Logan, UT, USA), which is adjusted with dextrose at 300 mOsm and 310 mOsm for internal and external solutions, respectively. For voltage clamp recordings, the cells are held at a holding potential of −60 mV (resting membrane potential), in where membrane currents are caused by voltage variation. The voltage shift is obtained using 10 mV voltage steps from −100 to 50 mV with duration of 800 ms, followed by a constant voltage of 20 mV during 200 ms. Thus, total membrane ionic current is recorded and the inward and outward currents are gave rise to a cell excitability and action potential generation.
For the current clamp experiments, the filters are open to 10 kHz and the pClamp software [43] generated square current pulses from −0.5 to 0.5 nA with 0.1 nA steps and 100 ms. The characteristics of the action potentials, caused by 1 ms suprathreshold pulses, are analyzed off-line using Clampfit (Molecular Devices) and Origin 8.0 (Microcal Software, Northampton, MA, USA). The phase-plane graphic of the cell voltage is obtained by plotting the first derivative of the membrane voltage (V) with respect to time (t) versus the membrane voltage [44]. The threshold of the action potential is the voltage at which whose dV/dt increases suddenly, and action potential amplitude is the voltage difference between the maximum peak and resting potential. The duration of the action potential at 50% is defined as the duration of the action potential measured at the half of its amplitude. The maximum depolarization and repolarization rate are obtained from the phase-plane graphic as the maximum and minimum dV/dt values, respectively. The after-hyperpolarization (AHP) is defined as the potential difference between the minimum voltage value after the action potential and the membrane potential preceding the action potential. All results are reported as the mean ± standard error. For all comparisons between the control glass and TiO 2 thin film surfaces a Student's t-test was used, considering as significant a p < 0.05.

Conclusions
TiO 2 thin films deposited by magnetron sputtering at room temperature and annealed with different temperatures (100, 300, 500, 800 and 1100 °C) were studied, analyzing their morphology and biocompatibility. The XRD patterns showed the transition of the anatase phase to rutile phase. To evaluate the biocompatibility in vitro of the TiO 2 thin films, primary cultures of dorsal root ganglia (DRG) neurons were performed, evaluating their survival rate and electrophysiological activity. As a biological system, DRG neurons were selected because their normal functional electrical activity in the materials can be parametrically defined, which is a clear indication of biocompatibility. The electrophysiological recordings showed that the DRG neurons can be maintained in culture during 24 h on TiO 2 thin films surfaces. The electrophysiological properties of the neurons, indicated the cells viability and showed that they are functionally similar to those deposited on the control substrate. In the TiO 2 thin films at 800 °C, the currents amplitudes were similar to those measured on control substrate. At this temperature, the TiO 2 thin films has morphological characteristics that could create an environment adequate for the cells due to the fact that they presented a normal set of membrane ionic channels. The result of these experiments confirmed the feasibility of using TiO 2 thin films as a biocompatible material for the stimulation and recording of electrical activity of DRG neurons in culture.
Future work will include the deposition of TiO 2 thin films on the microelectrodes surface and the readout circuit to fabricate CMOS-MEMS devices. This circuit will allow recording of other parameters of biological relevance such as micropotentials due to pH.

Author Contributions
Francisco López-Huerta, Agustín L. Herrera-May, Julián Hernández-Torres and Leandro García-González prepared and characterized the TiO 2 films; Blanca Cervantes and Octavio González realized the voltage clamp experiments; Blanca Cervantes, Rosario Vega and Enrique Soto analyzed the voltage clamp experiments and prepared the figures; Francisco López-Huerta, Agustín L. Herrera-May, Blanca Cervantes, Rosario Vega and Enrique Soto written and corrected the paper.