sensors

Article
Development and Characterization of Novel Conductive Sensing
Fibers for In Vivo Nerve Stimulation
Bertram Richter 1 , Zachary Mace 1,2 , Megan E. Hays 3 , Santosh Adhikari 3 , Huy Q. Pham 4 , Robert J. Sclabassi 1,2 ,
Benedict Kolber 5 , Saigopalakrishna S. Yerneni 6 , Phil Campbell 6 , Boyle Cheng 1 , Nestor Tomycz 1 ,
Donald M. Whiting 1 , Trung Q. Le 7, * , Toby L. Nelson 3 and Saadyah Averick 1, *

1 System Department of Neurosurgery, Allegheny Health Network, Pittsburgh, PA 15212, USA;

[email protected] (B.R.); [email protected] (Z.M.); [email protected] (R.J.S.); [email protected] (B.C.);
[email protected] (N.T.); [email protected] (D.M.W.)
2 Computational Diagnostics, Inc., Pittsburgh, PA 15213, USA
3 Department of Chemistry, Oklahoma State University, Stillwater, OK 74078, USA;
[email protected] (M.E.H.); [email protected] (S.A.); [email protected] (T.L.N.)
4 Department of Biomedical Engineering, North Dakota State University, Fargo, ND 58102, USA;
[email protected]
5 Department of Neuroscience, University of Texas at Dallas, Richardson, TX 75080, USA;
[email protected]
6 Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15217, USA;
[email protected] (S.S.Y.); [email protected] (P.C.)
7 Department of Industrial and Manufacturing Engineering, North Dakota State University,
Fargo, ND 58102, USA



* Correspondence: [email protected] (T.Q.L.); [email protected] (S.A.)



Citation: Richter, B.; Mace, Z.; Hays, Abstract: Advancements in electrode technologies to both stimulate and record the central nervous
M.E.; Adhikari, S.; Pham, H.Q.; system’s electrical activities are enabling significant improvements in both the understanding and
Sclabassi, R.J.; Kolber, B.; Yerneni, S.S.; treatment of different neurological diseases. However, the current neural recording and stimulating
Campbell, P.; Cheng, B.; et al. electrodes are metallic, requiring invasive and damaging methods to interface with neural tissue.
Development and Characterization of These electrodes may also degrade, resulting in additional invasive procedures. Furthermore, metal
Novel Conductive Sensing Fibers for
electrodes may cause nerve damage due to their inherent rigidity. This paper demonstrates that
In Vivo Nerve Stimulation. Sensors
novel electrically conductive organic fibers (ECFs) can be used for direct nerve stimulation. The ECFs
2021, 21, 7581. https://doi.org/
were prepared using a standard polyester material as the structural base, with a carbon nanotube ink
10.3390/s21227581
applied to the surface as the electrical conductor. We report on three experiments: the first one to
Academic Editor: David Niederseer
characterize the conductive properties of the ECFs; the second one to investigate the fiber cytotoxic
properties in vitro; and the third one to demonstrate the utility of the ECF for direct nerve stimulation
Received: 27 July 2021 in an in vivo rodent model.
Accepted: 7 November 2021
Published: 15 November 2021 Keywords: conductive sensing fiber; electrical probe; nerve stimulation

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in
published maps and institutional affil- 1. Introduction
iations.
Electrodes are used to stimulate and record electrical activity from the mammalian cen-
tral nervous system and have been widely applied in the medical field for the investigation
of spinal cord injuries, strokes, sensory deficits, and neurological disorders. Neuromodula-
tion, an evolving therapeutic approach based on the recording and delivery of electrical
Copyright: © 2021 by the authors. signals to targeted neurological sites, has been investigated for the treatment of condi-
Licensee MDPI, Basel, Switzerland. tions [1–8] such as Parkinson’s disease [9,10], dystonia, [11,12], tremor [13], psychiatric
This article is an open access article conditions [14], and pain disorders [15–18]. Most neural recording and stimulating elec-
distributed under the terms and trodes are metallic [19]. While metallic electrodes are highly efficient conductors of electric
conditions of the Creative Commons
charge, they present several disadvantages when interfacing with neural tissue [20–22].
Attribution (CC BY) license (https://
First, poor biocompatibility causes reactive gliotic changes in neural tissue [23–25]. Second,
creativecommons.org/licenses/by/
metals used as current injectors (e.g., platinum and iridium) are expensive and fragile.
4.0/).

Sensors 2021, 21, 7581. https://doi.org/10.3390/s21227581 https://www.mdpi.com/journal/sensors

Sensors 2021, 21, 7581 2 of 11

Third, the resistance inherent in metallic conductors causes energy to be dissipated as heat,
which can worsen reactive changes in the surrounding tissues [26–28], thus decreasing
electrode efficiency and potentially damaging biological tissue.
Next-generation electrodes are being designed to eliminate or minimize the above-
mentioned concerns without greatly affecting sensitivity [29–31]. Such electrodes are based
on material substrates with either an organic composition or a modified metallic struc-
ture [32,33]. These electrodes can both capture and deliver adequate electrical currents
while promoting neural integration with reduced tissue injury and inflammation [34–38].
Carbon fiber-based electrodes are one such alternative and have been shown to serve as
effective measurement and stimulation tools [39,40]. In some cases, carbon fibers have been
attached to silk fiber to reduce tissue damage, decrease electrode stiffness, and improve
recording stability [41]. Electrodes coated with nanoparticles have also shown promise for
neurological monitoring and treatment. In animal models, nanoparticle coating reduces
gliosis while improving contact impedance [42].
We have previously reported on electrically conductive organic fibers (ECFs) which
utilize a novel conductive ink [43] composed of single-walled nanotubes and regioregular
poly(3-hexylthiophene) stained on nonconductive cotton, polyester, or silk fibers. In our
previous paper, we both demonstrated that biosignals could be recorded with the stained
polyester fibers and reported on a number of the characteristics of the fibers. Furthermore,
the fibers have been utilized to fabricate the smart wearable multisensing textile-based
system for real-time prediction of disease onsets. The system is an extension of our previous
reported projects [44–46]. In this paper, we demonstrate that the polyester ECFs may be
used for electrical stimulation of neural tissue in an in vivo rodent model. In addition,
the electrical conductivity properties of the ECF and the cytotoxicity of the fiber cultured
in human cells are investigated. The organization of the paper is as follows: we first
describe the methods utilized for the fabrication of the ECFs and characterization of their
electrical properties; then, cytotoxicity studies; and finally, stimulation studies in an animal
model and statistical analysis. We report the results of these studies. The results are then
discussed, and our conclusions are summarized.

2. Methods
2.1. Fabrication of Electrically Conductive Organic Fibers (ECFs)
Fibers based on commercial polyester threads were prepared. Off-the-shelf polyester
threads (Joann Fabrics and Crafts) were coated with conductive ink without any additional
chemical treatment. To prepare the conductive ink, regioregular poly(3-hexylthiophene)
(rr-P3HT, 98% purity) was purchased from American Dye Source, Inc. (Montreal, QC,
Canada) and single-walled carbon nanotubes (SWCNTs ≥95% carbon basis ≥99%, 0.84 nm
average diameter) were purchased from Sigma Aldrich (St. Louis, MS, USA), and the
SWCNTs were added to the rr-P3HT solution in CHCl3, as previously described [43]. The
resulting mixture was ultrahigh sonicated using a Microson Ultrasonic Cell Disruptor for
30 min in an ice bath to avoid overheating and undesirable secondary reactions. A dipping
and drying technique was utilized to stain the polyester fibers with the conductive ink.
The process was repeated ten times per ECF. After staining, the fibers were oven dried for
15 min at 100 ◦ C.

2.2. Electrical Impedance

The electrical impedance of the fibers as a function of both frequency and length
were measured. In the first set of measurements, three polyester ECFs of the same length
(l = 2.5 cm) were obtained from one 7.5 cm fiber. The resistance of each fiber was measured
using a voltage-ohmmeter (VOM) (Fluke 87-V) and an impedance analyzer (Agilent 4395A)
at 1 MHz. In the second set of measurements, the impedance of each 2.5 cm ECF was
also measured over the 100 kHz–100 MHz (100,000 kHz) frequency range using the same
impedance analyzer. In the third set of measurements, the resistance (R) and reactance (X)
as a function of fiber length were measured. In these measurements, a 4 cm sample was
Sensors 2021, 21, 7581 3 of 11

cut from a 12 cm fiber thread and the resistance and reactance values were measured for
five trials with the impedance analyzer at 1 MHz. The 4 cm thread was then reduced in
length by 0.5 cm, and the measurements were repeated. This procedure was repeated to
the shortest length of 2 cm.

2.3. Cell Culture and Cytotoxicity

The loss of membrane integrity of three human cell types cultured with the ECFs was
investigated. Human embryonic kidney cells (HEK293; ATCC® CRL-1573™) (American
Tissue Culture Collection, Manassas, VA, USA), murine embryonic fibroblast cells (NIH3T3;
ATCC® CRL-1658™) (American Tissue Culture Collection, Manassas, VA, USA), and
human keratinocyte cells (HaCaT; #T0020001, AddexBio, San Diego, CA, USA) were grown
and maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Gaithersburg, MD,
USA), supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA,
USA) and 1% penicillin-streptomycin (Gibco, Gaithersburg, MD, USA). Then, 1 × 105 cells
were plated in the wells of three 12-well microplates (Corning Inc., Corning, NY, USA), one
for each cell type, and allowed to adhere overnight. Three 2 mm length polyester ECFs
were added to nine treatment wells for each microplate and co-incubated with cells for
72 h without media change. Three control (no ECF) wells were also analyzed for each cell
type. Post incubation, the ECFs were removed, and cells were labeled with CyQUANT® , a
nucleic acid-sensitive fluorescence assay (Thermo Fisher Scientific, Waltham, MA, USA).
Direct fluorescence intensities were measured with a spectrophotometer reader (TECAN,
Männedorf, Switzerland). Cytotoxicity was assessed by normalizing the fluorescence
intensities to the control group (no treatment) and plotted as percent viability.

2.4. Animal Model

The animal experiment was performed under the Allegheny General Hospital Insti-
tutional Animal Care and Use Committee approval (IACUC approval number 1036). A
male Sprague Dawley rat (110 weeks old, 1.5 kg) was utilized. The rat was anesthetized
with ketamine (90 mg/kg) (Mylan Pharmaceuticals, Morgantown, WV, USA). Maintenance
anesthesia was achieved using isoflurane (Piramal Critical Care Bethlehem, Pennsylvania,
USA) with 4% flow delivered via face mask. Thermoregulation was provided with a
heating pad. The depth of anesthesia was monitored with response to toe pinch stimulus
and bradypnea. The area over the left groin was clipped and prepped using iodine. Sharp
dissection was used to expose the neurovascular bundle in the groin. The sciatic nerve was
carefully separated from vascular structures with a sectioned vascular loop. Standard metal
subdermal needle electrodes (Ambu Neuroline subdermal needle electrode 12 × 0.40 mm,
Ballerup, Denmark) were utilized as described below. A metal ground electrode was placed
in the subcutaneous tissue of the left lower quadrant. For initial stimulation, a metal anode
electrode was placed medial to ground in the subcutaneous tissue of the left lower quadrant
and a metal cathode electrode was directly applied to the dissected sciatic nerve and hand
held in place. Metal recording electrodes were placed in the left tibialis anterior muscle in
bipolar montage to record the evoked compound muscle action potentials (CMAP). All
stimulation and recordings were performed using a NeuroNet VI system (Computational
Diagnostics Inc., Pittsburgh, PA, USA).
Stimulus frequency was set at 5.1 Hz and pulse width was set at 200 µs. The sciatic
nerve was stimulated with current starting at 0 mA and increased in 0.1 mA increments
until CMAPs were observed at 0.9 mA, and data were recorded at 1 mA. In the second
experiment, the metal cathode was replaced with a polyester ECF (l = 10 cm) by gently
wrapping the flexible ECF around the nerve, avoiding contact with any other tissue. The
stimulation parameters used previously were then repeated. The threshold was again
reached at 0.9 mA, and CMAPs were recorded at 1 mA. In the final experiment, the
metal anode electrode was replaced by a polyester suture-based ECF (l = 10 cm) sutured
directly into the fascia medial to the exposed sciatic nerve, resulting in a stimulating setup
consisting of an ECF anode and cathode. The previously described stimulation parameters
Sensors 2021, 21, 7581 4 of 11

were repeated with the following exception: the CMAP threshold was found at 2.1 mA
and CMAPs were subsequently recorded at a stimulus intensity of 2.2 mA, followed by
recording at 2.3 mA. For all ECF stimulation experiments, the coated polyester fibers
utilized as either stimulating anodes or cathodes were attached to Grass 10mm gold cup
electrode (Natus, Middleton, WI, USA) with Ten20 conductive paste (Weaver, Aurora, CO,
USA) to provide connection to the stimulator. The nerve was rested between experiments.
Upon completion of the study, the rat was euthanized using carbon dioxide at a rate of 40%
chamber volume per minute, followed by bilateral thoracotomy in a procedure approved
by the American Veterinary Medicine Association.

2.5. Statistical Analysis

All data were analyzed with either a Student’s t-test or analysis of variance (ANOVA),
followed by Tukey’s post hoc tests for multiple comparisons between treatment groups
and their controls using Prism 8 (GraphPad, San Diego, CA, USA). Statistical significance
was defined at p ≤ 0.05.

3. Results
3.1. Fiber Characterization
The polyester ECF fibers were observed with a light microscope, as shown in Figure 1.
Using vernier calipers, a fiber diameter of 200–250 µm was measured for each ECF.

Figure 1. Macro (left) and microscopic image (right) of polyester-based ECF.

3.2. Electrical Measurements of Polyester ECFs

The resistance and the resistive component of the impedance for each of the three
2.5 cm ECF samples measured as described in the Methods section are shown in Table 1.

Table 1. Resistance values obtained from three polyester ECFs, each of 2.5 cm length using a voltage-
ohmmeter (center column) and the impedance analyzer (right column).

ECF Sample Resistance (DC) Resistance (at 1 MHz)

1 6.40 kohms 6.48 kohms
2 5.16 kohms 5.19 kohms
3 5.35 kohms 5.35 kohms

In the second experiment, the resistive impedance of the polyester ECFs was measured
as a function of frequency over the range of 100 kHz–100 MHz. This frequency range was
chosen to fully capture the changes in the resistive impedance. As shown in Figure 2, when
the frequency applied to the sample exceeded 5 MHz (approximately at the −3 dB point),
the sample resistive impedance decreased at the rate of approximately 20 dB/dec.
Sensors 2021, 21, 7581 5 of 11

Figure 2. Resistance component of the impedance of three 2.5 cm long polyester samples taken from
the same 7.5 cm ECF, measured with the impedance analyzer. The resistance decreased as a function
of frequency above 5 MHz at a rate of approximately 20 dB/dec.

In the third experiment, the resistance and reactance values as a function of sample
length were measured for five trials with the impedance analyzer at a frequency of 1 MHz.
Figure 3 shows box plots of the resistance values of the polyester fibers as a function of the
sample length. The resistance increased as a function of the length of the polyester fibers.

Figure 3. Resistance values of the polyester ECFs measured at f = 1 MHz, five times, with respect to
the fiber length. Box plot with box of first and third quartile and median, and whiskers of minimum
and maximum values, neglecting outliers. Outliers are denoted as “+”.

Figure 4 shows the box plot of the reactance values of polyester fibers. This data
is consistent with a capacitive reactance with capacitance value of 88 picofarads at 4 cm
length and 270 picofarads at 2 cm length.

3.3. Fiber Cytotoxicity Testing

We found no statistically significant impact of incubation with coated ECFs in any of
the cell lines compared to the control wells (Figure 5), and all wells exhibited essentially
nearly 100% viability. These data suggest that in all three cell lines tested, there was
minimal toxicity from the ECFs in vitro for the time period tested.
Sensors 2021, 21, 7581 6 of 11

Figure 4. Reactance of polyester ECF measured at f = 1 MHz, five successive times, respectively, with
respect to the fiber length. Box plot with box of first and third quartile and median, and whiskers of
minimum and maximum values, not considering outliers. Outliers are denoted as “+”.

Figure 5. Effect of ECF fibers on the viability of HEK293, NIH3T3, and HaCaT cells. Cells were
incubated in the presence of 2 mm length ECF fibers, and the viability was determined by the direct
CyQUANT™ assay at 72 h. Cells grown on tissue culture plastic were used as the negative control
(no treatment group). Results are expressed as a percentage of negative control and bars indicate
mean ± SEM (n = 3 wells for each group), ns = not significantly different.

3.4. In Vivo Stimulation

We compared the capability of the ECFs to provide stimulation in vivo to stimulation
provided by a metal cathode and anode. The electrode was placed onto the nerve in the
similar manner to the fiber. The amount of steel and fiber was consistent across material
tested. The individual waveforms are presented in Figures 6 and 7. Both the steel and
polyester ECF were placed on top of the exposed nerve fiber. In the first experiment, the
sciatic nerve was stimulated with a metal cathode and a metal anode with current starting
at 0 mA and increased by 0.1 mA increments until a compound muscle action potential
(CMAP) was recorded by a pair of subdermal recording electrodes placed in the left tibialis
anterior muscle. The threshold was determined to be 0.9 mA and the CMAPs were then
recorded at 1 mA, as shown in Figure 6.
Sensors 2021, 21, 7581 7 of 11

Figure 6. Stimulation provided through metal electrodes with the cathode handheld against the
nerve, producing CMAPs recorded with subdermal needle electrodes at 1 mA stimulating current.
Eight successive CMAPS are overlaid.

In the second experiment, the metal cathode was replaced with an ECF. The fiber was
wrapped around the sciatic nerve, avoiding contact with any other tissue. The sciatic nerve
was again stimulated starting at 0 mA and increased by 0.1 mA increments until CMAPs
were obtained. The threshold was again reached at 0.9mA, with the CMAPs (Figure 7)
recorded at 1 mA stimulation.

Figure 7. Metal–ECF stimulation configuration of sciatic nerve. Panel (a) presents 10 successive CMAPs overlaid, obtained
by stimulation of the sciatic nerve using an ECF as a cathode at 1 mA. Panel (b) shows the ECF looped around sciatic nerve
propped up with a piece of nonconductive yellow tubing.
Sensors 2021, 21, 7581 8 of 11

In the third experiment, the metal anode was replaced by an ECF sutured directly into
the fascia medial to the exposed sciatic nerve. The sciatic nerve was again stimulated with
current starting at 0 mA and increased by 0.1 mA increments until CMAPs were observed
in the metal recording electrodes. In this case, the threshold was at 2.1 mA, and CMAPs
were recorded at 2.2 mA and 2.3 mA (Figure 8).

Figure 8. ECF–ECF stimulation configuration of sciatic nerve. Panel (a) presents 10 successive CMAPs overlaid, obtained by
stimulation of the sciatic nerve using an ECF as a cathode and anode at 2.3 mA. Panel (b) shows the sutured anodal ECF
(red arrow) adjacent to the sciatic nerve with the cathodal ECF looped around sciatic nerve, propped up with a piece of
nonconductive yellow tubing.

4. Discussions
In a previous study [43], we demonstrated that polyester ECFs could be used to record
bioelectric data, and we measured the resistance of the polyester ECFs as approximately
650 ohms per cm, while the electrode–skin impedances were measured to be approximately
between 18 and 38 kohms at 400 Hz. This study was designed to demonstrate that we
could also stimulate neural tissue using the polyester ECFs and to further define their
impedance characteristics, as well as demonstrate that they would have no cytotoxic effect
for short-term exposure to human cell lines.
The resistance (DC) and resistance impedance (at 1 MHz) values of the polyester ECF
were measured to be between 2140 and 2500 ohms/cm in three 2.5 cm samples. This was
about four times the values measured previously [43]. We believe the variances in the
resistance measurements are related to variations in the structure of the ECFs due to the
current fabrication process which does not control well for diameter of the coated fiber or
the degree of saturation of the fiber by the conductive ink.
The resistive component of the impedance for the same three samples referenced
above was measured between 100 kHz and 100 MHz. The values were approximately
constant out to approximately 5 MHz, and then decreased at 20 dB/dec. This demonstrates
that over the frequency range from 5 MHz to 100 MHz, it was increasingly easy for charge
to flow through the fibers. This is not easy to explain. However, this could be consistent
with the inverse of the well-known skin effect. At lower frequencies, the charge flows
along the surface of the ECFs, and as the frequency increases, the charge flows deeper
in the material. This is a new concept. These results suggest that we need to develop a
Sensors 2021, 21, 7581 9 of 11

deeper understanding of the chemical structure of these coated fibers and how electrons
are moved between the constituent molecules.
In the third resistance/impedance experiment, we measured the resistance and the
reactance of the ECF as a function of length at 1 MHz. Each measurement was repeated five
times on the same sample, demonstrating a high accuracy to the measurement technique.
The resistance increased as a function of length, as expected. The reactance was negative
and capacitive in nature, and also increased as a function of length going from 0.6 kohms at
2 cm in length to 1.8 kohms at 4 cm in length. This is consistent with capacitors connected
in series where the reciprocal of the equivalent capacitance equals the sum of the reciprocals
of the individual capacitances.
We also investigated the cytotoxicity of the polyester ECFs, in a 72 h experiment, using
three human cell lines. There was no cytotoxicity found over that period of time for those
particular cells. This does not fully answer the question of cytotoxicity or the possibility of
a gliotic reaction in vivo but gives us encouragement to move forward with more detailed
in vivo experiments.
Finally, we investigated the ability of the polyester ECFs to function as a stimulating
electrode. In these experiments, we demonstrated that the ECF could, indeed, be used to
stimulate neural tissue. The major distinction in these experiments is that the ECF–ECF
electrodes required 230% more current to produce an equivalent response, compared to the
experiments in which the electrodes were metal. This may be related to the higher resistance
impedance of the ECF compared to metal electrodes. These results again suggest that a
deeper understanding of the charge flow within these conductive ink-coated fibers is needed.

5. Conclusions
We have proposed a polyester-based organic conductive sensing fiber and the coating
process with the biocompatible conductive ink for in vivo neural tissue stimulation and
recording. We demonstrated that these fibers may perform both stimulation and recording.
Preliminary characterizations of resistance and impedance of the fibers as a function of
both length and frequency have been investigated. In another preliminary experiment,
we demonstrated that the polyester ECFs are not cytotoxic against three different human
cell types. Thus, it demonstrates the suitability of the fiber for further investigation into
its usefulness as interfaces to neural tissue. Future work will involve the development
of conductive materials for lead, wires, electrodes, and interconnections that enhance the
functionality, wearability, and reusability of electronics and biosensors.

Author Contributions: Conceptualization, T.L.N.; data curation, B.R., Z.M., H.Q.P., R.J.S., B.K., S.S.Y.,
P.C., D.M.W., B.C., N.T., T.Q.L. and S.A. (Saadyah Averick); formal analysis, B.R., Z.M., H.Q.P., S.S.Y.,
D.M.W., T.Q.L., S.A. (Saadyah Averick) and R.J.S.; funding acquisition, T.L.N.; R.J.S. Investigation,
B.R., Z.M., H.Q.P., S.S.Y., P.C., D.M.W., N.T., T.Q.L. and S.A. (Saadyah Averick); methodology, M.E.H.
and S.A. (Santosh Adhikari); resources, T.Q.L., T.L.N. and R.J.S.; supervision, N.T.; Validation, B.R.,
M.E.H., S.A. (Santosh Adhikari), H.Q.P., R.J.S., B.K., S.S.Y., P.C., D.M.W., B.C., N.T., T.Q.L. and
S.A. (Saadyah Averick); visualization, B.R., Z.M., H.Q.P., D.M.W., B.C., T.Q.L. and S.A. (Saadyah
Averick); writing—original draft, M.E.H. and T.L.N.; writing—review and editing, Z.M., T.Q.L.,
T.L.N., S.A. (Saadyah Averick) and R.J.S. All authors have read and agreed to the published version
of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The study was approved by the Allegheny General Hospital
Institutional Animal Care and Use Committee (IACUC approval number 1036).
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: SEA: B.R., B.C. and D.M.W. would like to thank the AHN Neuroscience institute
for support. Z.M. and R.J.S. were supported by CDI.
Conflicts of Interest: The authors declare no conflict of interest.
Sensors 2021, 21, 7581 10 of 11

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