Corrosion study of implanted TiN electrodes using excessive electrical stimulation in minipigs

: (1) Background: Titanium nitride (TiN) electrodes have been used for implantable stimulation and sensing electrodes for decades. Nevertheless, there still is a discrepancy between the in vitro and in vivo determined safe charge injection limits. This study investigated the consequences of pulsing implanted electrodes beyond the in vivo safe charge injection limits. (2) Methods: The electrodes were implanted for a month and then pulsed at 20 mA and 50 mA and 200 Hz and 400 Hz. Afterwards, the electrodes were investigated using electrochemical and analytical methods to evaluate whether electrode degradation had occurred. (3) Results: Electrochemical tests showed that electrodes that pulsed at 20 mA and 200 Hz (lowest electrical dose) had a signiﬁcantly lower charge injection capacity and higher impedance than the other used and unused electrodes. (4) Conclusions: The electrodes pulsed at the lowest electrical dose, for which no tissue damage was found, appeared to have degraded. Electrodes pulsed at higher electrical doses for which tissue damage did occur, on the other hand, show no signiﬁcant degradation in electrochemical tests compared to unused implanted and not implanted electrodes. It is thus clear that the tissue surrounding the electrode has an inﬂuence on the charge injection properties of the electrodes and vice versa.


Introduction
Titanium nitride (TiN) has been used for implantable electrodes for many decades, starting with cardiac pacing electrodes [1].The demands on cardiac pacing electrodes increased when it was desired to sense the heart rhythm, in order to provide rate-adaptive pacing [2].Despite the high voltages applied during cardiac pacing, the electrode polarization should remain low so that the heart signal can reliably be recorded [1,3].Porous electrodes were highly desirable for that purpose [4] but the electrodes should also be biocompatible [1] and corrosion resistant [3].
At the end of the previous century, TiN also received interest as a material for neural stimulation and recording electrodes [5].Neural stimulation and recording applications within this field include, among others, visual prosthesis [6], brain implants [7,8] and cochlear implants [9].Initially, studies reported conflicting results [5,6,9], which was likely due to differences in the fabrication method [4].The majority of studies, however, reported very favourable properties of porous TiN [5,6], which were due to its large surface area rather than specific material properties [4].
The performance of stimulation and recording electrodes can be evaluated using their safe charge injection limits (Q inj ), charge storage capacity (CSC) and impedance.Q inj is evaluated by comparing the electrode polarization under pulsing conditions to the safe potential limits established using slow sweep cyclic voltammetry (CV).The safe potential window is typically defined by the potentials at which water reduction and oxidation occurs.CSC is a measure of how much charge can be stored on the surface of the electrode and is measured using CV.The amount of charge available during fast pulsing, however, is typically much less than CSC.Impedance magnitude (typically at 1 kHz) can be used as a measure for battery consumption or recording performance.The lower the impedance, the better [10].
TiN has long been known as a biocompatible [29][30][31] and corrosion resistant material [3,32,33], even under cathodic high voltage pulsing conditions [34].Under anodic conditions, TiN oxidation reactions may occur, which primarily lead to passivation of these reactions until higher anodic voltages are reached [32].At very high anodic voltages, TiN will eventually be degraded [34].However, as Q inj is lower when implanted compared to in vitro [7,8,[21][22][23][24][25][26][27], unsafe voltages may be reached during pulsing.The aim of this study was therefore to investigate whether implanted TiN electrodes would degrade during pulsing when Q inj measured in vivo was exceeded but Q inj measured in saline was not.

Materials and Methods
Four Göttingen minipigs were implanted with four working electrodes (electrode pins) and four large surface area pseudo-reference disk electrodes.Minipigs were selected because the subcutaneous adipose tissue is similar to adipose tissue in humans.The number of electrodes and pseudo-reference electrodes was chosen in order not to cause excessive discomfort to the animals and thereby also to increase the homogeneity in the results.The electrodes were made from Ti6Al4V and coated with porous TiN.The animals recovered from anaesthesia and were monitored for one month before the corrosion experiments were conducted.The work was carried out according to Danish and European legislation (ethical approval license no: 2014-15-0201-00268).

Electrode Fabrication
TiN coatings were deposited on electrode pins (6 mm 2 ) made of a Ti6Al4V alloy and Ti disks (1000 mm 2 ) by reactive magnetron sputtering on a CC800/9 SiNOx coating unit (CemeCon AG, Würselen, Germany).The coatings were sputtered from four Ti targets (88 × 500 mm 2 ) with 99.5% purity in a Ar/N 2 mixture atmosphere.The purity of the gases was 99.999% and the Ar/N 2 -flow was 300 sccm/350 sccm.The deposition time was 21,000 s.The electrodes underwent three-fold rotation during the coating process.
The electrodes coatings were investigated after deposition using samples taken from the same batch.Scanning electron microscopy (SEM) (Nova 600, FEI Company, Hillsboro, OR, USA) images were taken at magnifications ranging from 450× to 25,000× to get an overview of the surface and to investigate the surface structure of the electrodes in detail.A silicium sample coated in the same process was used to study the thickness, homogeneity and porous structure of the TiN coating using SEM.Images were recorded at a magnification of 40,000×.
An ethylene tetrafluoroethylene (ETFE) coated 35N LT wire (Heraeus, Yverdon, Switzerland) was crimped to the hollow end of the electrode pins.A polyether ether ketone (PEEK) body and silicone tines were produced using injection moulding to insulate the electrode pins.The tines were first glued to the PEEK body using a silicone adhesive.The PEEK body with tines was then glued to the electrode pins also using a silicone adhesive.Further details and figures of the electrode production can be found in Reference [24].The electrodes were cleaned thoroughly before they were sterilized by an overdose of electron-beam processing.

Surgical Procedure
For electrode implantation, the animals were anesthetized using Propofol.Small incisions were made in the back and the electrodes were implanted using a custom-made implantation tool.This ensured that the electrodes were placed in tight pockets in subcutaneous adipose tissue, which promotes fast healing and ingrowth.Five electrodes were implanted into each pig, four of which were used for intense electrical stimulation and one electrode in each pig served as a control.Four counter electrode disks with a percutaneous wire were also implanted in each pig.The minipigs recovered from the procedure and were carefully monitored in order to detect and treat cases of infection.The electrodes were not used for one month until the pigs were anaesthetized again using sevoflurane to perform electrical stimulation.After the stimulation sessions were completed, the electrodes were carefully dissected from the tissue.The electrodes were extensively cleaned using demineralized water and alcohol, they were then rinsed and stored dry, so that they could be further investigated.

Electrical Stimulation
Electrical stimulation was performed using a DS5 (Digitimer, Hertfordshire, UK) per electrode.The device was shorted between the pulses using a custom-build set-up to prevent drifting of the baseline potential.Biphasic, charge balanced 200 µs square pulses were applied, cathodic first with an inter-phase interval of 40 µs during which no current was applied.Stimulation was performed for 6 h in total, divided into three 2-h sessions.Before, between and after these sessions, voltage transient measurements (VTM) were recorded for each electrode using a VersaSTAT 3 potentio-galvanostat (Princeton Applied Research, Oak Ridge, TN, USA).
Four stimulation paradigms were applied: During pilot experiments, it was verified that the group 1 stimulation paradigm did not cause tissue damage after one week of implantation (see Figure S1).To cause electrode damage, we decided to increase 2 parameters: stimulation frequency and stimulation amplitude.The stimulation frequency was doubled, which was expected to cause electrode damage due to an increasing trend in the inter-pulse potential [35].The stimulation amplitude was set to the maximum the DS5 can deliver, which was expected to cause electrode damage by increasing the electrode potential during stimulation.It was expected that group 2-4 protocols would result in tissue damage; therefore photographs were taken of the tissue surrounding the electrodes to document the amount of tissue damage.However, the focus of this study is corrosion and the electrodes were thus investigated more extensively using electrochemical and analytical methods.
During stimulation, the voltage transients were recorded every 30 min using an oscilloscope.From these voltage transients, the resistive drop after pulse cessation (IR-drop) was calculated as [10]: where E pulse_end is the recorded potential at the end of the cathodic pulse and E pulse_end+40 is the potential 40 µs after pulse cessation (see Figure 1) [10].E mc and E ma are the maximum cathodic and anodic voltage excursions after IR-drop is subtracted from the original voltage transient.VTM before, between and after the stimulation blocks, was performed using the VersaSTAT 3 potentio-galvanostat using the same stimulation pulse.
The pulsing capacitance (Cpulse) was computed using the slope (dV/dt) of the voltage transient: where Istim is the stimulation current (1 mA while implanted and 5 mA in the electrochemical characterization).Qinj was calculated using the current (Imax) at which Emc or Ema reached the safe potential limits (-0.6 and 0.9 V vs. open circuit potential, respectively) [10,24]: where t is the pulse duration (200 µs) and A is the geometrical surface area of the electrodes (6 mm 2 ).When voltage excursions exceeded machine limits (±10 V), Imax was extrapolated from the highest current assuming a linear relation.
where Vm and Im were the measured potential and current, respectively, and Vext and Iext were the extrapolated potential and current.When Vex reached the potential limits, Iext was used as Imax in (3).This method provided accurate results using data for which Imax was measured.

Coating Characterization
After explantation, all electrodes were characterized using SEM and energy-dispersive x-ray spectroscopy (EDX).The electrochemical properties were investigated using electrochemical impedance spectroscopy (EIS), CV and VTM.Two electrode groups were added to the 4 groups of active implants, therefore these measurements were performed on six electrode groups: • Group 1: implanted-20 mA, 200 Hz  VTM before, between and after the stimulation blocks, was performed using the VersaSTAT 3 potentio-galvanostat using the same stimulation pulse.
The pulsing capacitance (C pulse ) was computed using the slope (dV/dt) of the voltage transient: where I stim is the stimulation current (1 mA while implanted and 5 mA in the electrochemical characterization).Q inj was calculated using the current (I max ) at which E mc or E ma reached the safe potential limits (−0.6 and 0.9 V vs. open circuit potential, respectively) [10,24]: where t is the pulse duration (200 µs) and A is the geometrical surface area of the electrodes (6 mm 2 ).When voltage excursions exceeded machine limits (±10 V), I max was extrapolated from the highest current assuming a linear relation.
where V m and I m were the measured potential and current, respectively, and V ext and I ext were the extrapolated potential and current.When V ex reached the potential limits, I ext was used as I max in (3).This method provided accurate results using data for which I max was measured.

Coating Characterization
After explantation, all electrodes were characterized using SEM and energy-dispersive x-ray spectroscopy (EDX).The electrochemical properties were investigated using electrochemical impedance spectroscopy (EIS), CV and VTM.Two electrode groups were added to the 4 groups of active implants, therefore these measurements were performed on six electrode groups: : not implanted controls SEM (Nova 600, FEI Company, Hillsboro, OR, USA) images were recorded at magnifications varying from 450× to 10000× to obtain an overview of the surface and to investigate in detail the surface structure of the electrodes.SEM images of the not implanted control electrodes (group 6) were made, both to compare to the other electrode groups, as well as to investigate the uniformity of the coating after deposition.Further SEM analysis was carried out on a Si-wafer which was coated in the same process as the electrodes.The Si-wafer was mounted in a manner similar to the electrodes and the measured thickness is representative for the coating thickness on the electrodes.The advantage of using Si-wafer is that a cross-section analysis of the coating can be done easily.EDX (EDAX, AMETEK, Leicester, UK) spectra were made to investigate the chemical composition of the coatings after deposition and to determine whether the surface chemistry of the electrodes changed after having been implanted and after intense pulsing.
Electrochemical characterization measurements were performed in an electrochemical cell at room temperature using phosphate buffered saline as the electrolyte.The measurements were performed in a 3-electrode set-up, using the above mentioned porous TiN electrodes as working electrodes (0.06 cm 2 ), a Ag|AgCl reference electrode (1.6 cm 2 ) and a platinum foil counter electrode (50 cm 2 ).
Solartron, Model 1294 in conjunction with 1260 Impedance/gain-phase Analyzer (Solartron Analytical, Farnborough, UK) were used to perform EIS measurements.Accompanying SMaRT software was used to run the measurements.A sinusoidal current was used at frequencies from 0.1 Hz to 100 kHz, with 10 measurements per decade.Three different currents (5, 10 and 50 µA) were used to ensure that the measurement currents were in the linear operation range of the electrode [36].An integration time of 10 s was used to obtain a reliable and noise-free signal.
Cyclic voltammetry (CV) was performed by cycling the electrode potential was cycled between the safe potential limits (−0.6 and 0.9 V vs. Ag|AgCl) previously established for similar electrodes [24].The sweep rates used for CV were 0.05, 0.1, 0.5 and 1.0 V/s.Ten cycles were made at each sweep rate, the last cycle was used for data analysis.The cathodic charge storage capacity (CSC) was derived from the CV by taking the integral of the CV below the zero-current axis [10].
VTM were conducted in the same manner as described above for the implanted electrodes, except the 3-electrode setup and the electrochemical cell were employed.The maximum charge injection limit (Q inj ) and pulsing capacitance (C pulse ) were derived according to Equations ( 1)-(3).

Statistics
The data recorded during the 2-h pulsing sessions using an oscilloscope were filtered using a low-pass Butterworth filter (passband 5 kHz, stopband 15 kHz).E mc and IR-drop were then normalized to the first measurement (session 1, start).E mc was selected for statistical analysis to represent the electrode polarization and IR drop , as a measure of the tissue resistance.Before, between and after the pulsing sessions, voltage transients were recorded using the VersaSTAT 3 (Princeton Applied Research, Oak Ridge, TN, USA).From these voltage transients Q inj and C pulse were used to further quantify the electrochemical performance of the electrodes.A linear mixed model was used to statistically analyse the data.Parameters (group, session, time and combinations thereof) were added stepwise to the model, until adding another parameter did not make a significant difference to the model.
One-way ANOVA was used to investigate the electrochemical properties of the electrodes after explantation (electrochemical cell setup).The following electrochemical properties of the 6 different electrode groups were used for statistical analysis:

•
Cathodic CSC at 0.05 and 1.0 V/s • Impedance magnitude at 0.1 Hz Significant findings are reported at p-values smaller than 0.05.

Results
All implantations were carried out without any complications.The animals recovered well from the surgery and no infections were observed during the month the electrodes were implanted.

General Coating Characteristics
In contrast to the well-known yellow-golden coloured TiN, the coatings on the electrodes had a brownish colour.To analyse the structure and chemical composition of the coating, the electrodes were studied in SEM and EDX.The overview SEM image (Figure 2a) shows a uniform coating on the electrode and in the corresponding EDX spectrum (Figure 2b) the expected peaks belonging to Ti and N are present.The quantification of the amounts of Ti and N from an EDX spectrum is difficult because the K-line of N and the L-line of Ti are very close.Here, numbers close to a 1:1 atomic ratio of Ti to N are found (note that weight-% is used in Figure 2b).The SEM images in Figure 3a clearly show a faceted structure, typical for TiN deposited at high pressure.The cross-section SEM image in Figure 3b shows the porous morphology of the coating as well.The coating thickness is approximately 6 µm.

Results
All implantations were carried out without any complications.The animals recovered well from the surgery and no infections were observed during the month the electrodes were implanted.

General Coating Characteristics
In contrast to the well-known yellow-golden coloured TiN, the coatings on the electrodes had a brownish colour.To analyse the structure and chemical composition of the coating, the electrodes were studied in SEM and EDX.The overview SEM image (Figure 2a) shows a uniform coating on the electrode and in the corresponding EDX spectrum (Figure 2b) the expected peaks belonging to Ti and N are present.The quantification of the amounts of Ti and N from an EDX spectrum is difficult because the K-line of N and the L-line of Ti are very close.Here, numbers close to a 1:1 atomic ratio of Ti to N are found (note that weight-% is used in Figure 2b).The SEM images in Figure 3a clearly show a faceted structure, typical for TiN deposited at high pressure.The cross-section SEM image in Figure 3b shows the porous morphology of the coating as well.The coating thickness is approximately 6 µm.

Results
All implantations were carried out without any complications.The animals recovered well from the surgery and no infections were observed during the month the electrodes were implanted.

General Coating Characteristics
In contrast to the well-known yellow-golden coloured TiN, the coatings on the electrodes had a brownish colour.To analyse the structure and chemical composition of the coating, the electrodes were studied in SEM and EDX.The overview SEM image (Figure 2a) shows a uniform coating on the electrode and in the corresponding EDX spectrum (Figure 2b) the expected peaks belonging to Ti and N are present.The quantification of the amounts of Ti and N from an EDX spectrum is difficult because the K-line of N and the L-line of Ti are very close.Here, numbers close to a 1:1 atomic ratio of Ti to N are found (note that weight-% is used in Figure 2b).The SEM images in Figure 3a clearly show a faceted structure, typical for TiN deposited at high pressure.The cross-section SEM image in Figure 3b shows the porous morphology of the coating as well.The coating thickness is approximately 6 µm.

Changes in Electrochemical Properties during Intensive Pulsing
The shorting part of the setup broke down during the last series of measurements.The last two stimulation sessions could therefore not be completed with one of the electrodes in the 20 mA-00 Hz group.The data obtained with this electrode after the breakdown was not used in the analyses.
The significant parameters of the statistical model for IR-drop were: Time, Session, Group × Session and Time × Session.For E mc , Group was an additional significant parameter of the statistical model.Figure 4 shows that the results for IR-drop and E mc were similar.Both IR-drop and E mc were significantly larger during session 1 compared to sessions 2 and 3 for electrode groups 2, 3 and 4. IR-drop and E mc were only significantly larger during session 1 compared to sessions 2 and 3 at the 30 and 60 min measurements.Figure 4a,b also show that IR-drop (for groups 2, 3 and 4) and E mc (all groups) were significantly larger after 30 and 60 min of pulsing compared to after 90 and 120 min of pulsing during session 1.
The shorting part of the setup broke down during the last series of measurements.The two stimulation sessions could therefore not be completed with one of the electrodes in the 20 mA-00 Hz group.The data obtained with this electrode after the breakdown was not used in the analyses.
The significant parameters of the statistical model for IR-drop were: Time, Session, Group × Session and Time × Session.For Emc, Group was an additional significant parameter of the statistical model.Figure 4 shows that the results for IR-drop and Emc were similar.Both IR-drop and Emc were significantly larger during session 1 compared to sessions 2 and 3 for electrode groups 2, 3 and 4. IRdrop and Emc were only significantly larger during session 1 compared to sessions 2 and 3 at the 30 and 60 min measurements.Figure 4 A and B also show that IR-drop (for groups 2, 3 and 4) and Emc (all groups) were significantly larger after 30 and 60 min of pulsing compared to after 90 and 120 min of pulsing during session 1.
Emc of group 1 electrodes was significantly larger than Emc of group 2, 3 and 4 electrodes in all sessions.During session 2, Emc of group 2 electrodes was significantly smaller than Emc of group 1 and 3 electrodes and during session 3 Emc of group 2 electrodes was significantly smaller than Emc of group 1, 3 and 4 electrodes.Figure 4 C and D show that IR-drop and Emc of all electrode groups was significantly smaller at the start of stimulation compared to after 30, 60, 90 and 120 min of pulsing during session 3. The same was found for session 2.  , an increase in IR-drop was seen from the start of stimulation, after which IR-drop remained stable.The IR-drop of group 2, 3 and 4 electrodes was significantly smaller during sessions 2 and 3 compared to session 1. (c) The same trend was observed for E mc of all electrode groups but to an even greater extent (notice the axis).(d) An increase in E mc was also observed from the start of stimulation during sessions 2 and 3 (shown).E mc of group 2, 3 and 4 electrodes was also significantly smaller during sessions 2 and 3 compared to session 1 but notice again the difference in the axis of IR-drop and E mc .
E mc of group 1 electrodes was significantly larger than E mc of group 2, 3 and 4 electrodes in all sessions.During session 2, E mc of group 2 electrodes was significantly smaller than E mc of group 1 and 3 electrodes and during session 3 E mc of group 2 electrodes was significantly smaller than E mc of group 1, 3 and 4 electrodes.Figure 4c,d show that IR-drop and E mc of all electrode groups was significantly smaller at the start of stimulation compared to after 30, 60, 90 and 120 min of pulsing during session 3. The same was found for session 2.

Changes in Electrochemical Properties between Pulsing Sessions
For both C pulse and Q inj , the significant fixed effects were: Time, Group and Time*Group.The results of the statistical analysis for C pulse and Q inj were identical, except for a baseline difference between electrode groups observed for Q inj (p = 0.045).Q inj of group 2 electrodes (8.3 ± 2.4 µC/cm 2 ) was significantly smaller than Q inj of group 3 and 4 electrodes (15.8 ± 2.4 µC/cm 2 ).
Figure 5a,b show that Q inj and C pulse , respectively, of group 1 electrodes did not change significantly.

Changes in Electrochemical Properties between Pulsing Sessions
For both Cpulse and Qinj, the significant fixed effects were: Time, Group and Time*Group.The results of the statistical analysis for Cpulse and Qinj were identical, except for a baseline difference between electrode groups observed for Qinj (p = 0.045).Qinj of group 2 electrodes (8.3 ± 2.4 µC/cm 2 ) was significantly smaller than Qinj of group 3 and 4 electrodes (15.8 ± 2.4 µC/cm 2 ).
Electrode group 1 had a significantly smaller Qinj than all other electrode groups after the first pulsing session, which remained after the second and third pulsing session.Furthermore, electrode group 2 had a significantly smaller Qinj than electrode groups 3 and 4 after the first pulsing session.This difference also remained significant after pulsing sessions 2 and 3.The same group differences were observed for Cpulse.

Electrochemical Characteristics after Explantation
The results of the electrochemical characterization in phosphate buffered saline were largely consistent across measurements, as shown in Figure 6.Group 1 electrodes had a significantly smaller CSC at 0.05 and 1.0 V/s and a significantly larger impedance magnitude at 0.1 Hz compared to all Electrode group 1 had a significantly smaller Q inj than all other electrode groups after the first pulsing session, which remained after the second and third pulsing session.Furthermore, electrode group 2 had a significantly smaller Q inj than electrode groups 3 and 4 after the first pulsing session.This difference also remained significant after pulsing sessions 2 and 3.The same group differences were observed for C pulse .

Electrochemical Characteristics after Explantation
The results of the electrochemical characterization in phosphate buffered saline were largely consistent across measurements, as shown in Figure 6.Group 1 electrodes had a significantly smaller CSC at 0.05 and 1.0 V/s and a significantly larger impedance magnitude at 0.1 Hz compared to all other electrode groups.They also had a significantly smaller Q inj compared to group 2 electrodes and the control electrodes in groups 5 and 6.But the Q inj of group 1 electrodes was not significantly different from group 3 and 4 electrodes.

Coating properties after Explantation
SEM images (see Figure 7) showed that the electrode surfaces were intact after 6 h of intense stimulation.The coatings were all undamaged and had the same faceted structure, which is typical for TiN, as the electrodes had after deposition of the coating.Figure 7 indicates that there were no differences in chemical composition of group 1 and group 4 electrodes.The EDX spectra of all electrodes in all groups showed similar levels of titanium, nitrogen and oxide.

Coating Properties after Explantation
SEM images (see Figure 7) showed that the electrode surfaces were intact after 6 h of intense stimulation.The coatings were all undamaged and had the same faceted structure, which is typical for TiN, as the electrodes had after deposition of the coating.Figure 7 indicates that there were no differences in chemical composition of group 1 and group 4 electrodes.The EDX spectra of all electrodes in all groups showed similar levels of titanium, nitrogen and oxide.

Discussion
Several previous studies [37][38][39] have shown that TiN coatings grown at high N-partial pressures become porous.These coatings have a brown colour that clearly distinguish them from the standard golden-coloured TiN used on tools and components.For use in electrode applications, it is essential to use the porous type of TiN coating as they have a high effective surface area, which in turn leads to a low impedance [3,4].Cunha et al. [39] did a systematic study of the influence of Ncontent of the morphological structural and electrochemical properties of TiN coatings.In the case of high N-content (Ti:N ratio 1:1.34), these authors obtain results similar to ours regarding the coating morphology.The reason for the discrepancy in chemical composition (in our case we measure a Ti:N ratio 1:1 with EDX) could be that Cunha et al. [39] have determined the chemical composition using Rutherford Backscattering Spectrometry, which provides a more precise determination compared to EDX.Other studies have found that porous near-stoichiometric TiN coatings can be obtained just by adjusting the energy available during film growth [40].
Six groups of electrodes were used in this study, five of which were implanted and four of which were used for 6 h of intense pulsing at different electric doses: • Group 1: 20 mA, 200 Hz • Group 2: 20 mA, 400 Hz • Group 3: 50 mA, 200 Hz

Discussion
Several previous studies [37][38][39] have shown that TiN coatings grown at high N-partial pressures become porous.These coatings have a brown colour that clearly distinguish them from the standard golden-coloured TiN used on tools and components.For use in electrode applications, it is essential to use the porous type of TiN coating as they have a high effective surface area, which in turn leads to a low impedance [3,4].Cunha et al. [39] did a systematic study of the influence of N-content of the morphological structural and electrochemical properties of TiN coatings.In the case of high N-content (Ti:N ratio 1:1.34), these authors obtain results similar to ours regarding the coating morphology.The reason for the discrepancy in chemical composition (in our case we measure a Ti:N ratio 1:1 with EDX) could be that Cunha et al. [39] have determined the chemical composition using Rutherford Backscattering Spectrometry, which provides a more precise determination compared to EDX.Other studies have found that porous near-stoichiometric TiN coatings can be obtained just by adjusting the energy available during film growth [40].
Six groups of electrodes were used in this study, five of which were implanted and four of which were used for 6 h of intense pulsing at different electric doses: During intense pulsing, IR-drop was derived from the voltage transients as a measure of tissue impedance and E mc was used as a measure of electrode polarization.Between the three 2-h pulsing sessions, Q inj was evaluated and from these voltage transients C pulse was derived.
No significant changes were observed during intense pulsing for group 1 electrodes, receiving the lowest electrical dose.For group 2, 3 and 4 electrodes the IR-drop and E mc decreased and Q inj and C pulse increased.The stability in pulsing properties of group 1 electrodes together with the fact that these electrodes received the lowest electrical dose, would intuitively lead to the expectation that these electrodes did not corrode [4, 7,8,[23][24][25][26].For group 2, 3 and 4 electrodes, on the other hand, it could be expected that corrosion may have occurred, even though the observed electrochemical changes are favourable in the light of pulsing capability (higher charge injection, lower electrode polarization).Passivation at high anodic potentials was the main expected corrosive reaction [33,34], leading to decreased pulsing capability.Excessive bubbling due to water reduction [34], however, may lead to cracking of the coating.This could increase the surface area and thereby lead to an apparent increase Q inj .The results of the electrochemical characterizations after explantation showed that group 1 electrodes had significantly deteriorated electrochemical properties compared to all other electrode groups.Group 2, 3 and 4 electrodes, on the other hand, had no different electrochemical properties after explantation than the two control groups (5 and 6).
Analytical investigations could neither confirm nor reject the electrochemical results.SEM images show that all coatings seemed to be intact.EDX spectra did not reveal differences between the harshest and mildest stimulated electrodes either.However, it must be noted that it is difficult to distinguish between the oxygen and nitrogen signal using EDX because the K-peaks of the two elements are quite close.As oxidation of the coating could have occurred, other analytical methods have been attempted (X-ray photoelectron spectroscopy and Time-of-Flight Secondary Ion Mass Spectrometry) in order to detect any differences in oxygen amounts.Preliminary results were unsuccessful, mainly because the geometry and size of the electrode is quite challenging in both the experimental set-ups.It is, however, very unlikely that the coating oxidized without showing any signs of damage.Norlin et al. [34] show SEM images porous TiN electrodes after anodic pulsing, which are severely damaged.For the current electrodes no signs of damage were found using SEM and EDX analysis.
Corrosion studies of stimulation electrodes have been performed extensively in saline [3,34,[41][42][43].Some studies found that the damage threshold is exactly at the water window limit [43], others suggest that the water window limit may be too conservative under pulsing conditions [41,44] and yet others suggest that corrosion may occur even within the limits of water reduction and oxidation [42].TiN has been most extensively investigated by Norlin et al. [3,34,45].Their pulsing study was carried out using 700V pulses of both anodic and cathodic polarity [34].As expected, TiN showed severe corrosion upon anodic pulsing but was stable when cathodic pulses were applied.The voltages recorded in this study during constant current pulsing were, however, a factor 15-30 smaller.In a later study, the electrodes were aged using more conservative voltages (−3 and 1 V vs. Ag|AgCl), corresponding to 4 months of use based on the charge passed [3].TiN proved very stable, which was expected, as high anodic voltages were avoided.In the current study, very high anodic voltages were also avoided by using a cathodic first stimulation paradigm.Anodic voltages between 1 and 3 V (vs.open circuit potential) were observed with the highest voltages in the 50 mA groups (groups 3 and 4).No signs of corrosion were observed for the electrodes in those groups, while corrosion of the anodically pulsed samples by Norlin et al. [34] was obvious in SEM images.
It has also been shown before that safe limits obtained in inorganic solutions do not necessarily apply to electrodes in protein containing solution [46] and implanted electrodes [47,48].It was therefore concluded that proteins must protect the electrode surface against corrosion [46,48,49].However, Q inj was never measured in vivo for these electrodes and it is therefore unclear whether or not it was exceeded [47,48].The in vitro water window limits for platinum were not exceeded in either of the studies but in both studies corrosion was observed nevertheless [47,48].Shepherd et al. [48] argue, however, that corrosion was not stimulation-induced but due to production failures.In the current study, Q inj (measured in vivo) was exceeded for all stimulated electrode groups (1, 2, 3 and 4) during all stimulation sessions.However, group 1 and 2 electrodes were pulsed at approximately 20% of Q inj as measured in saline and groups 3 and 4 electrodes were pulsed at approximately 50% of the in vitro Q inj .Robblee et al. [47] stimulated their electrodes at approximately 5% and 30% of Q inj in vitro.They observed platinum dissolution for all electrodes pulsed at 30% of Q inj (in vitro) and less for electrodes pulsed at 5%.Shepherd et al. [48] stimulated the electrodes at 5-10% of Q inj in vitro but concluded that the observed corrosion was not stimulation-induced.This makes it obvious that in vitro safe limits cannot be applied in vivo.However, it does not rule out that limits measured in vivo using techniques developed in vitro may be too conservative.
Interestingly, we found that corrosion most likely occurred for the electrodes pulsed at the lowest electrical dose (group 1 electrodes; 20% of Q inj in vitro and 200 Hz).As group 2, 3 and 4 electrodes showed no signs of corrosion in the electrochemical characterizations after explantation, the occurrence of corrosion seems not only dependent on the electrode potentials or charge delivered.We suspect that the occurrence of corrosion is not only medium dependent (organic vs. inorganic, basic vs. acidic) but also tissue dependent.Although tissue damage was not the focus of this study, it seems to play an important role.No tissue damage seems to have occurred for electrode group 1, while tissue damage with increasing severity occurred for electrode groups 2-4 (see Figure S2).The lack of tissue damage for electrode group 1 is confirmed by the lack of change in IR-drop, which is representative of tissue impedance [10].The same amount of charge density per phase was injected for electrode groups 1 and 2 but due to the increased frequency tissue damage is likely to have occurred in group 2 [50,51].Tissue damage obviously occurred in electrode groups 3 and 4 (see Figure S2).Based on the IR-drop data, it appears that the tissue was damaged during the first hour of the first pulsing session for electrode groups 2, 3 and 4. It seems that a new electrode-tissue interface was formed that remained during pulsing session 2 and 3.This new electrode-tissue interface allowed for more charge injection, as Q inj and C pulse were significantly increased after the first pulsing session compared to before pulsing.And although the electrodes were still pulsed beyond the increased Q inj in vivo, the formation of a new electrode-tissue interface and corresponding increase in Q inj may thus have prevented corrosion.
The electrode-tissue interface appears to play a very important role with regards to the occurrence of corrosion.These results can therefore only be applied to stimulation electrodes implanted in adipose tissue, like ours [23,24,52] and like Bion [53] for example.They cannot be applied to implants in the brain [47], the cochlea [48] or the blood stream [1,2,4].Furthermore, our electrode is a macro-electrode (0.06 cm 2 ).There are indications that different charge injection limits apply to smaller microelectrodes [51].These results might therefore not apply to microelectrodes.Lastly, as it is challenging to work with larger animals, such as minipigs, the number of animals is low compared to rodent studies for example.The results, however, are consistent across measurements and rather homogeneous within the electrode groups and were thus statistically significant.
With the recent increase in investment in "electroceuticals," the development of novel, smaller and more sophisticated implants may be anticipated [54].It is therefore more important than ever before to establish safe limits that apply to these specific implants [51].We show that this is not only relevant in the light of tissue damage but also with respect to corrosion and long-term electrochemical performance of the implants.In the light of tissue damage due to corrosion, TiN appears to be a very suitable material for implants.There seems to occur no dissolution of the material [55], like with Pt [46] and IrOx [43].As long as very high anodic potentials are avoided [34], we show that no corrosion occurs even after almost 9 million pulses.When corrosion does occur, its product (a passivation layer) remains attached to the electrodes and is not harmful to the tissue [33,55].

Conclusions
It was long suspected that in vitro safe limits established for implantable electrodes may not be applicable in vivo, which we confirm here.We also show that the type of tissue in which the electrode is implanted has an influence on safety limits.Biocompatibility and corrosion resistance cannot be viewed as two separate properties of implantable stimulation (and sensing) electrodes.Tissue responses influence the electrochemical behaviour of implanted electrodes and use of the electrodes influences the tissue surrounding the electrodes.It is therefore of great importance that safe limits are established for each electrode depending on the tissue in which it will be implanted.

Supplementary Materials:
The following are available online at http://www.mdpi.com/2075-4701/9/4/389/s1, Figure S1: (a) Tissue around the tip of an electrode stimulated at 20 mA-200 Hz after 1 week of implantation.The tissue was stained using haematoxylin and eosin (H&E), which is the most commonly applied stain in medical diagnostics.Some inflammatory cells can still be observed but capsule formation has begun to take place.(b) Cells around the silicone part the electrode appear very similar to those around the electrode tip, indicating no signs of stimulation-induced tissue damage.Figure S2: (a) The tissue around electrodes in group 1 showed no signs of tissue damage upon sacrifice.(b) The tissue around electrodes in group 2 showed some redness around the electrode tip, which likely is due to tissue damage.(c) The tissue around the electrode tips of electrodes in group 3 showed obvious tissue damage but the tissue around the insulated parts was unaffected.(d) The tissue around the electrode tips of electrodes in group 4 showed even more extensive tissue damage and bleeding.Nevertheless, the tissue around the insulated parts was unaffected.
Funding: This research was funded by the Danish National Advanced Technology Foundation.

Figure 1 .
Figure 1.IR-drop, dV/dt, Emc and Ema are derived from the original data, while in the manuscript normalized data are presented.The data is normalized by subtracting the IR-drop and setting the prepulse potential to 0.

Figure 1 .
Figure 1.IR-drop, dV/dt, E mc and E ma are derived from the original data, while in the manuscript normalized data are presented.The data is normalized by subtracting the IR-drop and setting the pre-pulse potential to 0.

Figure 2 .
Figure 2. (a) Scanning electron microscope (SEM) image of the coated electrode.The indicated region is the area over which EDX was performed.The scale-bar is 500 µm.(b) Typical energy dispersive Xray spectroscopy (EDX) spectrum corresponding to the area indicated in (a).

Figure 2 .
Figure 2. (a) Scanning electron microscope (SEM) image of the coated electrode.The indicated region is the area over which EDX was performed.The scale-bar is 500 µm.(b) Typical energy dispersive X-ray spectroscopy (EDX) spectrum corresponding to the area indicated in (a).

Figure 2 .
Figure 2. (a) Scanning electron microscope (SEM) image of the coated electrode.The indicated region is the area over which EDX was performed.The scale-bar is 500 µm.(b) Typical energy dispersive Xray spectroscopy (EDX) spectrum corresponding to the area indicated in (a).

Figure 4 .
Figure 4. (a) IR-drop increased from baseline and then decreased during the first pulsing session for electrode groups 2, 3 and 4. (b) During session 2 and 3 (shown), an increase in IR-drop was seen from the start of stimulation, after which IR-drop remained stable.The IR-drop of group 2, 3 and 4 electrodes was significantly smaller during sessions 2 and 3 compared to session 1. (c) The same trend was observed for Emc of all electrode groups but to an even greater extent (notice the axis).(d) An increase in Emc was also observed from the start of stimulation during sessions 2 and 3 (shown).Emc of group 2, 3 and 4 electrodes was also significantly smaller during sessions 2 and 3 compared to session 1 but notice again the difference in the axis of IR-drop and Emc.

Figure 4 .
Figure 4. (a) IR-drop increased from baseline and then decreased during the first pulsing session for electrode groups 2, 3 and 4. (b) During session 2 and 3 (shown), an increase in IR-drop was seen from the start of stimulation, after which IR-drop remained stable.The IR-drop of group 2, 3 and 4 electrodes was significantly smaller during sessions 2 and 3 compared to session 1.(c)The same trend was observed for E mc of all electrode groups but to an even greater extent (notice the axis).(d) An increase in E mc was also observed from the start of stimulation during sessions 2 and 3 (shown).E mc of group 2, 3 and 4 electrodes was also significantly smaller during sessions 2 and 3 compared to session 1 but notice again the difference in the axis of IR-drop and E mc .

Figure 5 .
Figure 5. (a) After the first pulsing session Qinj was increased compared to before pulsing for electrode groups 2, 3 and 4. (b) The same was observed for Cpulse of electrode groups 2, 3 and 4. (c) Normalized voltage transients recorded before pulsing at 3 mA.(d) Normalized voltage transients recorded at 3 mA after 2 h of pulsing.

Figure 5 .
Figure 5. (a) After the first pulsing session Q inj was increased compared to before pulsing for electrode groups 2, 3 and 4. (b) The same was observed for C pulse of electrode groups 2, 3 and 4. (c) Normalized voltage transients recorded before pulsing at 3 mA.(d) Normalized voltage transients recorded at 3 mA after 2 h of pulsing.

Metals 2018, 8 ,
x FOR PEER REVIEW 9 of 16 other electrode groups.They also had a significantly smaller Qinj compared to group 2 electrodes and the control electrodes in groups 5 and 6.But the Qinj of group 1 electrodes was not significantly different from group 3 and 4 electrodes.

Figure 6 .
Figure 6.(a) The impedance magnitude was significantly larger for electrode group 1 compared to the other electrode groups, which was implanted and pulsed at the lowest electrical dose.(b) The cyclic voltammogram shows that the charge storage capacity of electrode group 1 was significantly smaller than the other electrode groups.(c) The normalized voltage transients at 5 mA show that the slope of the group 1 electrodes was larger than the slopes of the other electrode groups but no significant difference was found for Cpulse.(d) Qinj of group 1 electrodes was significantly smaller than Qinj of group 2, 5 and 6 electrodes.

Figure 6 .
Figure 6.(a) The impedance magnitude was significantly larger for electrode group 1 compared to the other electrode groups, which was implanted and pulsed at the lowest electrical dose.(b) The cyclic voltammogram shows that the charge storage capacity of electrode group 1 was significantly smaller than the other electrode groups.(c) The normalized voltage transients at 5 mA show that the slope of the group 1 electrodes was larger than the slopes of the other electrode groups but no significant difference was found for C pulse .(d) Q inj of group 1 electrodes was significantly smaller than Q inj of group 2, 5 and 6 electrodes.

Figure 7 .
Figure 7. (a) Close-up SEM image (magnification: 10,000×) of an electrode in group 1 (20 mA-200 Hz), showing that the surface structure is still intact (scale bar: 10 µm).The same is true for (b), showing the detailed structure of an electrode in group 4 (50 mA-400 Hz, scale bar: 10 µm, magnification: 15 000×).(c) Overview SEM (magnification 450×) and corresponding EDX images of an electrode in group 1 show that the chemical composition of the electrode surface after pulsing was very similar to (d) the chemical composition of the electrode surface of an electrode in group 4. Also visualized by an overview SEM (magnification 450×) and corresponding EDX images of an electrode in group 4.

Figure 7 .
Figure 7. (a) Close-up SEM image (magnification: 10,000×) of an electrode in group 1 (20 mA-200 Hz), showing that the surface structure is still intact (scale bar: 10 µm).The same is true for (b), showing the detailed structure of an electrode in group 4 (50 mA-400 Hz, scale bar: 10 µm, magnification: 15,000×).(c) Overview SEM (magnification 450×) and corresponding EDX images of an electrode in group 1 show that the chemical composition of the electrode surface after pulsing was very similar to (d) the chemical composition of the electrode surface of an electrode in group 4. Also visualized by an overview SEM (magnification 450×) and corresponding EDX images of an electrode in group 4.