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Biomaterials 28 (2007) 513–523

www.elsevier.com/locate/biomaterials

The effect of polypyrrole with incorporated neurotrophin-3 on thepromotion of neurite outgrowth from auditory neurons

Rachael T. Richardsona,b,�, Brianna Thompsonc, Simon Moultonc, Carrie Newbolda,May Ghee Luma, Adrian Camerona, Gordon Wallacec, Robert Kapsaa,d,

Graeme Clarka, Stephen O’Learya,b

aThe Bionic Ear Institute, 384 Albert Street, East Melbourne, Vic. 3002, AustraliabThe University of Melbourne, Department of Otolaryngology, Royal Victorian Eye and Ear Hospital, East Melbourne, Vic., Australia

cThe Intelligent Polymer Research Institute, University of Wollongong, NSW, AustraliadResearch Centre for Neuroscience and Neurology Research, St. Vincent’s Hospital, Fitzroy, Vic., Australia

Received 27 June 2006; accepted 8 September 2006

Available online 27 September 2006

Abstract

This research aims to improve the nerve–electrode interface of the cochlear implant using polymer technology to encourage neuron

survival, elongation and adhesion to the electrodes. Polypyrrole (Ppy) doped with p-toluene sulphonate (pTS) is an electroactive polymer

into which neurotrophin-3 (NT3) can be incorporated. Ppy/pTS7NT3 was synthesised over gold electrodes and used as a surface for

auditory neuron explant culture. Neurite outgrowth from explants grown on Ppy/pTS was equivalent to tissue culture plastic but

improved with the incorporation of NT3 (Ppy/pTS/NT3). Electrical stimulation of Ppy/pTS/NT3 with a biphasic current pulse, as used

in cochlear implants, significantly improved neurite outgrowth from explants. Using 125I-NT3, it was shown that low levels of NT3

passively diffused from Ppy/pTS/NT3 during normal incubation and that electrical stimulation enhanced the release of biologically

active NT3 in quantities adequate for neuron survival. Furthermore, Ppy/pTS/NT3 and its constituents were not toxic to auditory

neurons and the Ppy/pTS/NT3 coating on gold electrodes did not alter impedance. If applied to the cochlear implant, Ppy/pTS/NT3 will

provide a biocompatible, low-impedance substrate for storage and release of NT3 to help protect auditory neurons from degradation

after sensorineural hearing loss and encourage neurite outgrowth towards the electrodes.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Controlled drug release; Electroactive polymer; Electrical stimulation; Nerve growth factor; Nerve regeneration

1. Introduction

Sensorineural hearing loss occurs after loss or damage ofcochlear sensory hair cells. The cochlear implant restoreshearing to the profoundly deaf by directly stimulatingauditory neurons (spiral ganglion neurons; SGNs) with anarray of electrodes inserted into the cochlea. A secondaryeffect of hair cell loss is the gradual degeneration andapoptosis of SGNs due to an absence of hair cell-mediatedneurotrophic support [1]. Loss of neurotrophic support for

e front matter r 2006 Elsevier Ltd. All rights reserved.

omaterials.2006.09.008

ing author. The Bionic Ear Institute, 384 Albert Street,

e, Vic. 3002, Australia. Tel.: +613 9929 8281;

1958.

ess: rrichardson@bionicear.org (R.T. Richardson).

SGNs may reduce the effectiveness of the cochlear implant,which is currently the only therapy available for profoundsensorineural hearing loss. Administration of exogenousneurotrophins such as neurotrophin-3 (NT3) or brain-derived neurotrophic factor (BDNF) to the cochleapreserves SGNs after aminoglycoside-induced experimen-tal deafness [2–7]. Resprouting of nerve fibres alsoaccompanies SGN survival following neurotrophic factortreatment [2]. The fibres often have abnormal trajectoriessuggesting that neurotrophins may need to be deliveredmore locally or with greater control. Since neurotrophinsurvival effects are short-lived [8], long-term slow-releasesystems are being explored for SGN protection andresprouting around the time of implantation for animproved nerve–electrode interface.

ARTICLE IN PRESSR.T. Richardson et al. / Biomaterials 28 (2007) 513–523514

Osmotic pumps for the delivery of neurotrophins to thecochlea, as demonstrated in guinea pigs [9], pose asignificant and unacceptable risk of infection for humanuse. Alternatively, the cochlear round window membraneallows diffusion of some types of drugs into the cochlea,including neurotrophins [10], but is less reliable inproviding consistent drug doses [11]. In a further emergingoption, coating of the cochlear implant with a neurotro-phin-eluting biopolymer stands to more effectively andsafely protect SGNs and encourage nerve fibre growthtowards the cochlear implant electrodes during and afterimplantation. This study considers a polymer known aspolypyrrole (Ppy) to perform these functions.

Ppy is a polymer that conducts electricity based on itsredox state. It is an excellent substrate for cell adhesion[12–18] and apart from minor inflammatory tissue reac-tions is well tolerated when implanted in the peripheralnerve, brain or abdominal cavity [14,19]. Anions (dopants)catalyse Ppy polymerisation and provide a means forvarying the physical and biological properties of Ppy [20].For example, heparin improves hydrophilicity and sup-ports the adhesion of endothelial cells to Ppy [15], whiledoping with laminin peptide fragments encourages moreneural tissue growth than plain electrodes [21]. Large orstrongly charged Ppy dopants tend to be strongly boundand do not diffuse away after long periods of incubation insolution [21]. Other incorporated dopants can be moremobile within the polymer due to their smaller size, weakercharge or greater solubility in the receiving solution. Theycan subsequently be released from Ppy with appliedelectrical stimulation [22].

For coating cochlear implant electrodes, Ppy with astrongly charged p-toluene sulphonate (pTS) dopant hasappropriate structural and mechanical properties andallows the subsequent incorporation of NT3 [23]. We haveshown that NT3 can be released from Ppy/pTS/NT3 withelectrical stimulation in quantities that might provebeneficial for SGN survival after sensorineural hearing lossto the potential advantage of cochlear implant technology[23]. Approximately 93 ng/cm2 NT3 was incorporated intoPpy/pTS with the release rate controllable by the type ofapplied electrical stimulation. It is already known that sometypes of electrical stimulation are beneficial for the healthand survival of neurons in vitro and in vivo; a constantpotential of 100mV or a constant current of 10mAenhanced differentiation and increased neurite length inneuron-like pheochromocytoma cells (PC12 cells) grown onPpy doped with polystyrene [19]. Likewise in the cochlea,combining neurotrophic factors and electrical stimulationfrom the cochlear implant provides greater benefit toneuron survival after deafness than either of the treatmentsalone [24]. If regenerating SGN processes are provided withneurotrophic factors, electrical stimulation and a biocom-patible surface on which to grow, they might be encouragedto grow closer to or onto electrodes of new generationcochlear implants for a more integrated nerve–electrodeinterface during the early post-implantation period.

This paper examines whether a conductive Ppy/pTS/NT3 coating of electrodes can support and extend auditoryneuron survival and neurite outgrowth through NT3release. Postnatal rat SGN explants were used to assessSGN interaction and response to Ppy/pTS/NT3 with orwithout electrical stimulation, focussing on neurite out-growth, biocompatibility and cell adhesion. Finally,electrode impedance was analysed to determine thesuitability of Ppy/pTS/NT3 as a coating for cochlearimplant electrodes.

2. Materials and methods

2.1. Polymer synthesis

Ppy was synthesised in disposable eight-well culture slides with each

well containing a circular 0.05mm2 sputter-coated gold working electrode

and a 21.8mm2 common gold return electrode (Applied BioPhysics). The

working electrode area was increased by removing the insulating layer

with a short 20% NaOH incubation followed by rinses in Milli-Q water.

The new working electrode size was 18mm2 in 6 of the wells and 21.3mm2

in two of the wells, the difference arising from the shape of the lead wire

(Fig. 1a). Polymerisation was carried out in aqueous solution containing

0.2M distilled pyrrole (Merck), 0.05M analytical grade pTS (Aldrich) and

where specified, 2mg/ml NT3 (Chemicon International). All polymers were

grown galvanostatically using the central gold electrode as the working

electrode and a stainless steel mesh electrode placed in each well as the

auxiliary/reference electrode. Ppy/pTS was polymerised at a current

density of 2mA/cm2 for 90 s, before the addition of 2 mg/ml NT3 (where

required) and a further 60min growth (two-layered approach). After

polymerisation, wells were washed five times with Milli-Q water and

stored at 4 1C in sterile Milli-Q water until use.

2.2. Culture slide preparation

In experiments specifying a cell adhesion molecule (CAM) layer, the

Ppy-coated culture slides or equivalent eight-well plastic culture slides

(Lab-Tek, Nunc) were incubated in 0.5mg/ml poly-ornithine (Sigma;

diluted in 0.15M borate buffer pH8.56) at 4 1C overnight. On the day of

culture, the poly-ornithine was removed and the culture slides washed

three times with Hepes-buffered Eagle’s media (HEM). Culture slides

were then incubated in 0.01mg/ml laminin (Gibco; diluted in HEM)

at 37 1C for at least 2 h. Prior to plating of explants, the laminin

was removed, the culture slides were rinsed once in HEM and then filled

with 300ml Dulbecco’s modified Eagle’s media (DMEM)/Sato culture

media (modified from Bottenstein and Sato [25]; DMEM containing

0.01% bovine serum albumin (BSA) fraction V, 100mg/ml transferrin,

16mg/ml putrescine, 10mg/ml insulin, 0.4 mg/ml thyroxine, 0.3mg/ml

thyronine, 0.06mg/ml progesterone, 0.04mg/ml selenium, 2mM glutamine

and 4.5 g/l glucose). Culture slides that were not coated with CAMs

were subjected to the same incubations and procedures, but in CAM-free

solutions.

2.3. Animals

Time-mated pregnant Albino-Wistar rats were purchased from the

University of Adelaide. Rat pups were used at post-natal days 4–6.

National Institutes of Health (NIH) guidelines for the care and use of

laboratory animals (NIH Publication #85-23 Rev. 1985) were observed.

The Animal Research Ethics Committee of the Royal Victorian Eye and

Ear Hospital approved the care and use of the animals in this study (ethics

#03/096A).

ARTICLE IN PRESS

1 2 3 4

5 6 7 8

COMMON

+1 mA

-1 mA

Active electrode

20 µs

100 µs

3.78 ms short

60

40

20

-20

-40

-60

-5e-5 5e-5 1e-4 2e-4 2e-40

Time (s)

Voltage (V)

Vt

Va

Vp

(a)

(b)

(c)

Fig. 1. Electrode culture slide for Ppy polymerisation, explant culture and

electrical stimulation. (a) Eight-well culture slides consisted of 18–21mm2

thin-film gold circular active electrodes and 22mm2 gold return electrodes.

(b) The current waveform applied to the electrodes had71mA amplitude,

100ms pulse width, 20ms open-circuit interphase gap and 3.78ms short-

circuit phase. (c) An example of the resulting voltage waveform when

biphasic current pulses were applied to gold electrodes. Impedances were

calculated from the voltage waveform using peak voltage output (Vt) as

well as components of Vt (Va and Vp) which characterise the initial rapid

voltage drop and the remaining voltage drop, respectively.

R.T. Richardson et al. / Biomaterials 28 (2007) 513–523 515

2.4. Dissection and explant culture

Post-natal day 4–6 rat pups were anaesthetised on ice before

decapitation. Under aseptic conditions, the skulls were opened mid-

sagittally and the brain removed. The remaining tissue was cut into two

sagittal halves and placed in HEM containing antibiotics. Under a

dissecting microscope, cochleae were removed from the temporal bones

and stripped of their otic capsule, stria vascularis and organ of Corti using

forceps. The upper basal turn was isolated from the rest of the cochlea

using a scalpel. SGNs were separated from the inner modiolus, cut into

four or five pieces and placed in the prepared culture slides. Where

indicated, NT3 (Chemicon; prepared as a stock solution in phosphate

buffered saline (PBS) containing 1% BSA and then diluted to a working

concentration in DMEM/Sato) was added to the culture media at

20–80 ng/ml to make a final culture volume of 300ml. The 0 ng/ml NT3

control consisted of PBS/1% BSA diluted exactly as per NT3 in DMEM/

Sato. Cultures were placed in a 37 1C humidified incubator containing

10% CO2 for 3 days prior to fixing and immunocytochemistry.

2.5. Electrical stimulation

Explants were grown for 24 h to allow settling and adhesion. Culture

slides were then attached to a pulse generator and oscilloscope via an in-

house connector. Charged-balanced biphasic current pulses at 250Hz were

applied for 1 h. The waveform had71mA current amplitude, 100ms pulsewidth, 20ms open-circuit interphase gap and 3.78ms short-circuit phase

between pulses (Fig. 1b). Following stimulation, explants were grown for a

further 3 days prior to fixing and immunocytochemistry.

2.6. Fixation and immunocytochemistry

SGN explant cultures were fixed in ice-cold 100% methanol for 15min

and rinsed once in PBS. Blocking buffer (PBS/3% foetal calf serum) was

applied for 1 h at room temperature and then replaced with anti-

neurofilament 200 kD primary antibody (Chemicon) diluted 1:2000 in

blocking buffer. Incubation was for 1 h at room temperature. Explants

were washed 3 times with PBS and incubated for 1 h at room temperature

in FITC-conjugated anti-rabbit secondary antibody (Molecular Probes),

diluted 1:200 in blocking buffer. Following 3 rinses in PBS, an anti-fade

mounting medium containing 40,6-diamidino-2-phenylindole (DAPI;

Vector Laboratories) was applied and the explants viewed via fluorescent

microscopy.

2.7. Measurement of neurite length and number

Explants were photographed on a Zeiss Axioplan fluorescent micro-

scope. Blinded counts of the number of neurites were conducted at the

point where they exited the explant. Explants without visible neurites were

excluded from final analyses to ensure that only tissue capable of growing

neurites was dissected. Neurite length was measured as the linear distance

from point of exit to the end of the longest branch of that neurite. Length

measurements of 10 neurites were taken from each explant, or all neurites

if there were less than 10 neurites present. The measurements were made at

regular intervals around the circumference of the explant.

2.8. Scanning electron microscopy (SEM)

Explants grown on Ppy/pTS or Ppy/pTS/NT3 were fixed in 4% (w/v)

paraformaldehyde for 15min and then exposed to increasing concentra-

tions of ethanol for 30min each (30%, 50%, 70%, 90% and 100%).

Samples were placed in a Balzers CPD-030 critical point dryer before

being sputter coated in gold (Edwards S150B gold sputter coater). Images

were taken on a Philips XL30-FEG field emission scanning electron

microscope (School of Botany, University of Melbourne, Australia).

2.9. Measurement of NT3 release from Ppy/pTS/NT3

Ppy/pTS/NT3 was polymerised on 1 cm2 gold-Mylar (CPFilms Inc.) as

described, substituting NT3 for 125I-NT3 (ProSearch International).

Resulting polymers were coated with CAMs, placed in 1ml DMEM/Sato

and subjected to the same conditions as the electrical stimulation study

(albeit at room temperature instead of 37 1C). DMEM/Sato samples were

taken after each day of incubation and immediately following electrical

stimulation (25 h timepoint). Samples were analysed on a Cobra-5000 gcounter and corrected for the decay rate of 125I. The masses of 125I-NT3

incorporated and released from the polymer were calculated using the

specific activity of the radio-labelled 125I-NT3.

2.10. Impedance measurements

Impedances of gold, Ppy/pTS-coated gold or Ppy/pTS/NT3-coated

gold electrodes were measured according to a previously described method

[26]. Briefly, a single charge-balanced current pulse (1.5mA amplitude (i),

50ms pulse width, 25ms interphase gap) was presented across the working

ARTICLE IN PRESSR.T. Richardson et al. / Biomaterials 28 (2007) 513–523516

and reference electrodes using an in-house optically isolated, battery

operated biphasic current pulse stimulator. The voltage waveform was

recorded using an analogue oscilloscope (Gould, DSO 400) and electrode

impedance was calculated using Ohm’s law (Fig. 1c).

2.11. Statistical analyses

Neurite outgrowth and length data for the validation study were

transformed to a normal distribution by taking the square root or the

natural logarithm, respectively, due to a positive skew. One-way analysis

of variation (ANOVA) was performed on the transformed data, followed

by a Bonferroni multiple comparison procedure. Untransformed data

from the growth of auditory neurons on Ppy was analysed using three-way

ANOVA, while data from electrical stimulation and NT3 release studies

were analysed using one-way ANOVA followed by a multiple comparison

procedure (Bonferroni). Confidence for all data analyses was set at 95%

(po0:05).

3. Results

3.1. Validation of explant assay for examining neurite

outgrowth on Ppy

SGN explants were used to measure biological activity ofNT3 in Ppy substrates in three main studies; validation ofneurite outgrowth from explants on tissue culture plastic,evaluation of neurite outgrowth on Ppy and an electricalstimulation study. A total of 795 explants were analysed.Some explants had no neurites, either because NT3 was notsupplied or not effective or due to the occasional dissectionof tissue lacking SGNs. Since these cases could not bedistinguished, 150 explants without neurites (19%) wereexcluded from analysis. Table 1 shows the number explantsanalysed and the percentage of explants excluded pergroup.

Table 1

Number of experiments, wells and explants for all groups and percentage of e

Material CAM NT3 No. of expts No. of wells

Plastic � 0 2 9

� 40 5 15

+ 0 9 17

+ 20 5 12

+ 40 9 20

+ 60 6 18

+ 80 3 8

Ppy/pTS � 0 4 7

� 40 3 7

+ 0 13 54

+ 40 6 21

Ppy/pTS/NT3 � 0 3 4

� 40 3 5

+ 0 14 56

+ 40 2 4

Ppy/pTS +stim + 0 2 10

Ppy/pTS/NT3 +stim + 0 2 11

aNumber of explants remaining after fixation and staining, including explan

Explants grown on plastic coated with CAMs (lamininand poly-ornithine) consisted of a mass of inner cochleartissue containing the cell bodies of SGNs but devoid of haircells. Neurite outgrowth was quantified and is defined inthis paper as the number of neurites exiting the cellularmass. Neurite outgrowth was unrelated to explant size,with smaller explants sometimes having more neurites thanlarger explants and vice-versa. There were 6.2571.2neurites per explant in the absence of NT3 after 3 days inculture. Addition of 20, 40, 60 or 80 ng/ml NT3 increasedneurite outgrowth up to 3-fold (Figs. 2 and 3a). Neuritelength was also affected by the concentration of NT3added to the culture media, peaking at 40 ng/ml NT3 (Fig.3b). The differences in means among groups for both SGNcounts and neurite lengths were greater than expected bychance (po0:05), while pair-wise comparisons showed astatistically significant difference between 0 and 40 ng/mlNT3 for neurite number and neurite length (po0:05). Forall remaining assays, only neurite number was assessedusing 0 or 40 ng/ml NT3.

3.2. Neurite outgrowth from explants grown on Ppy

The following experiments compared neurite outgrowthfrom explants grown on plastic, Ppy/pTS or Ppy/pTS/NT37CAMs and 740 ng/ml NT3. There were significanteffects of substrate (plastic, Ppy/pTS or Ppt/pTS/NT3),CAM coatings and addition of NT3 to the culture mediaon neurite outgrowth from explants, but no interactionsbetween these groups. Explants grown on CAM-coatedsurfaces displayed 2.3 fold greater neurite outgrowthcompared to explants grown on uncoated surfaces(po0:0001; Fig. 4). Similarly, addition of NT3 to theculture media enhanced neurite outgrowth from explants

xplants excluded from the final analysis due to an absence of neurites

No. of explantsa Explants per wella Explants excluded (%)

17 1.9 58.8

43 2.9 27.9

46 2.7 30.4

20 1.7 25.0

47 2.4 6.4

21 1.2 33.3

9 1.1 0.0

9 1.3 44.4

17 2.4 29.4

175 3.2 23.4

71 3.4 26.8

11 2.8 9.1

8 1.6 0.0

193 3.4 12.4

17 4.3 11.8

47 4.7 6.4

44 4.0 0.0

ts without neurites.

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Fig. 2. NT3-induced neurite outgrowth from SGN explants. Representa-

tive images of SGN explants grown on CAM-coated tissue culture plastic

with various concentrations of NT3 added to the culture media. SGN

neurites were visualised by immunocytochemistry with a neurofilament-

200 primary antibody and a fluorescent secondary antibody. In the

absence of NT3, very few neurites were observed from explants (a), while

explants grown in 20 ng/ml NT3 (b), 40 ng/ml NT3 (c) and 60 ng/ml NT3

(d) demonstrated increased numbers of sprouting neurites. Scale bars are

100mm.

Fig. 3. Validation of explant assay. SGN explants were plated onto plastic

tissue culture slides coated with CAMs and cultured in media containing

0–80ng/ml NT3. (a) The number of neurites growing out from SGN

explants was counted proximal to the explant edge for each condition.

There were 6.2571.2 neurites per explant in the absence of NT3. The

addition of NT3 stimulated increased neurite outgrowth from SGN

explants. (b) Neurite length exhibited a dose relationship to NT3 that

peaked at 40 ng/ml NT3. Error bars represent standard error of the mean

(�po0:05, ��po0:001 compared to the 0 ng/ml NT3 control).

R.T. Richardson et al. / Biomaterials 28 (2007) 513–523 517

1.5 fold (po0:01; Fig. 4). The Ppy/pTS/NT3 surfaceyielded explants with 1.7 fold greater neurite outgrowthcompared to plastic or Ppy/pTS (po0:002; Fig. 4) whilethere was no difference between neurite outgrowth fromexplants growing on Ppy/pTS and plastic surfaces. Fig. 5shows neurite outgrowth from explants in all conditionstested in which it can be observed that Ppy/pTS/NT3,regardless of CAMs and NT3, enhanced neurite outgrowthfrom explants.

Explants growing close to but not directly on top ofPpy/pTS/NT3 also showed increased neurite outgrowth(20.579.2 neurites per explant) compared to those grownnear Ppy/pTS (9.471.0 neurites per explant) and compar-able neurite outgrowth to explants grown directly on Ppy/pTS/NT3 (19.172.2 neurites per explant).

3.3. Neural and cellular interactions with Ppy

Interactions of neurons and other cells with Ppy/pTSand Ppy/pTS/NT3 were examined by SEM. The surface ofPpy/pTS consisted of raised, uneven globules and incor-poration of NT3 made no detectable difference to thismorphology. Under low power, explants appeared asa central mass of tissue with cells radiating outwards(Fig. 6a). A closer inspection of the cells revealed thatmany were fibroblasts adhering to raised globules of Ppy(Fig. 6b). Interspersed with the fibroblasts was a mixtureof other cells including osteoblasts and Schwann cells.Neurites radiated from explants as long fibres, exhibitingbranching and fasciculation and did not appear influenced

by surface topography (Fig. 6c). Fibroblasts and neuritesappeared to contact other cells and Ppy equally, with nopreference for one or the other (Fig. 6d). Growth coneswith extending filopodia were visible on the growing endsof many neurites (Fig. 6e).

3.4. Electrical stimulation of Ppy-coated electrodes

The electrical stimulus used to release NT3 from Ppy/pTS/NT3 was based on cochlear implant stimulationparameters (see Fig. 1b). Given the area of electrodes usedin this study, this stimulus is predicted to result in a chargedensity of 0.47–0.55 mC/cm2 (geometric). In the absence ofelectrical stimulation, explants plated on Ppy/pTS grew16.272.6 neurites per explant. Explants grown under thesame conditions but with 1 hr of stimulation sprouted19.371.7 neurites per explant. This was not statisticallydifferent from unstimulated explants (p ¼ 1:0). Explants

ARTICLE IN PRESS

Fig. 4. Neurite outgrowth is affected by surface chemistry. SGN explants were grown on Ppy/pTS or Ppy/pTS/NT3, with or without a coating of CAMs

and in media containing 0 or 40 ng/ml NT3. (a) Explants grown on uncoated Ppy/pTS exhibited poor neurite outgrowth. (b) The addition of a CAM

coating to Ppy/pTS enhanced neurite outgrowth, as did the inclusion of NT3 in the culture media (c). Compared to Ppy/pTS, neurite outgrowth was also

improved by incorporating NT3 into the Ppy, exemplified by this explant, grown on Ppy/pTS/NT3 (d). The addition of a CAM coating to Ppy/pTS/NT3

(e) and NT3 to the media (f) made further dramatic differences to neurite outgrowth. Scale bars are 100 mm.

Fig. 5. The addition of a CAM coating, NT3 to the culture media and the

incorporation of NT3 into Ppy enhance neurite outgrowth from explants.

Explants were grown on plastic, Ppy/pTS or Ppy/pTS/NT3 with or

without a CAM coating and in the presence or absence of 40 ng/ml NT3 in

the culture media. The number of SGNs sprouting from each explant was

counted. Growth of explants on Ppy/pTS/NT3 enhanced neurite out-

growth compared to explants grown on tissue culture plastic or Ppy/pTS

(po0:002). In addition, the presence of NT3 in the culture media or the

addition of CAMs to plastic, Ppy/pTS or Ppy/pTS/NT3 also resulted in

greater neurite outgrowth compared to the respective controls (po0:01).Error bars represent standard error of the mean. ‘Plas’ refers to tissue

culture plastic, Ppy refers to Ppy/pTS and Ppy/NT3 refers to Ppy/pTS/

NT3.

R.T. Richardson et al. / Biomaterials 28 (2007) 513–523518

grown on Ppy/pTS/NT3 without electrical stimulation hadon average 28.372.4 neurites per explant. In agreementwith results from Fig. 5, this was a significantly greaternumber of neurites per explant compared to Ppy/pTS(po0:05). One hour of electrical stimulation of Ppy/pTS/NT3 increased the number of neurites per explant to

42.073.0, a significantly greater number than unstimulatedPpy/pTS/NT3 and all other means (po0:001) (Fig. 7a–c).

3.5. NT3 release

Six Ppy/pTS/125I-NT3 polymers were incubated insimilar conditions to the stimulation study with halfreceiving 1 h of electrical stimulation between the 24and 25 h timepoints. In the first 24 h of incubation,polymers in stimulated and unstimulated groups releaseda similar amount of NT3 (2.270.1 and 2.1701 ng,respectively). Subsequent stimulation of Ppy/pTS/125I-NT3 for 1 h resulted in the release of a further1.070.018 ng NT3 while unstimulated Ppy/pTS/125I-NT3released only 0.4770.010 ng NT3 during the same timeperiod. The release rate over the following days did notdiffer between the two groups. Cumulative data showsstimulated polymers releasing more NT3 over 4 days ofincubation compared to unstimulated polymers (po0:05;Fig. 7d).

3.6. Toxicity tests

In the event that small amounts of pTS or pyrrole leachout of the polymer during normal incubation or electricalstimulation, we conducted preliminary toxicity tests of pTSor monomeric pyrrole to auditory neurons. Explants weregrown on plastic surfaces coated with CAMs. Pyrrole(diluted between 1:100 and 1:10,000) and 0.05–50mM pTSwere added to the culture medium containing 40 ng/mlNT3 for 3 days. There was no difference between neuriteoutgrowth from explants cultured in the highest and lowestconcentrations of pyrrole (26.370.67 versus 30.0710.0neurites per explant, n ¼ 3, p ¼ 0:7). Similar results werefound for the highest and lowest concentrations of pTS

ARTICLE IN PRESS

Fig. 6. Neural interactions with Ppy. SEM images of explants grown on Ppy/pTS (a, b, d) or Ppy/pTS/NT3 (c, e). Under low power, explants appear as

cells and neurites radiating from a central mass of tissue (a). With higher magnification, the raised nodules of Ppy/pTS are clearly visible. A fibroblast

growing on this Ppy/pTS surface appears as a flat cell adhering to raised sections of Ppy/pTS (b). A neurite extending from an explant (out of view on the

left side of the picture) grows alongside another neurite in a fascicle (arrowhead) and then becomes a single neuron. A nerve branch is visible (arrow) (c).

Closer to the explant, nerve fibres can grow very densely, interacting with both the Ppy and other cells (d). Growth cones are often visible at the growing

tip of neurites; in this case the growth cone terminates on a raised Ppy/pTS/NT3 nodule and is extending a filopodium (arrow) (e).

R.T. Richardson et al. / Biomaterials 28 (2007) 513–523 519

(35.8 77.5 versus 32.3711.7 neurites per explant, n ¼ 4,p ¼ 0:8).

3.7. Impedance measurements

The impedances of Ppy/pTS and Ppy/pTS/NT3-coatedgold electrodes were compared to uncoated gold electrodes.Three impedance values were derived from the resultingvoltage waveforms; total impedance (Zt), access resistance(Ra; relating to changes in electrolyte) and polarisationimpedance (Zp; relating to changes at the electrode surface)(Fig. 8 inset). There was no difference between the threeimpedance values for Ppy/pTS- and Ppy/pTS/NT3-coatedgold electrodes compared to uncoated gold electrodes(Fig. 8).

4. Discussion

4.1. Validation of explant assay

Dissociated SGN culture has been previously used toscreen neurotrophic factors for their potential to promoteauditory neuron survival and neurite outgrowth for in vivo

applications [27–32]. However, dissociated SGN cultureprovides an unrealistic environment for neurite outgrowthas it limits the neuron-supporting cell and neuron–neuroninteractions that normally occur in vivo. SGN explantassays, used previously to examine neurite interaction withdifferent surfaces [33,34], were employed here to promote asupportive cellular environment around the neurons morereflective of that found in the cochlea. The SGN explantassay was also chosen to mimic auditory neuron outgrowthin vivo, where increased numbers of peripheral dendriteshave been observed to grow out from cochlear tissue afterneurotrophic factor treatment [2–4].Neurite outgrowth from explants showed a similar dose-

relationship to NT3 as dissociated SGN cultures [28,35].Both assays demonstrated some SGN survival in theabsence of NT3, although it must be remembered thatexplants without neurites were excluded from our analysis.When all data was analysed, including explants withoutneurites, explants still averaged 3.071.4 neurites in theabsence of NT3. The 1% BSA in the NT3 and controlsolutions, the non-neural supporting cells within theexplant and other media components could contributeto this baseline survival. Neurite outgrowth implies the

ARTICLE IN PRESS

Fig. 7. Electrical stimulation of Ppy for release of NT3. Explants were

grown for 24 h on CAM-coated Ppy/pTS or Ppy/pTS/NT3 and subjected

to a biphasic current pulse stimulus for 1 h. Neurite outgrowth was

examined after a further 3 days in culture. A greater number of neurites

per explant were observed on explants grown on the stimulated Ppy/pTS/

NT3 (a) compared to explants grown on stimulated Ppy/pTS (b). (c)

Stimulation of Ppy/pTS did not significantly alter the number of neurites

per explant compared to unstimulated Ppy/pTS (p ¼ 1:0). On the other

hand, explants grown on Ppy/pTS/NT3 with applied stimulation had

enhanced outgrowth of neurites compared to explants grown on

unstimulated Ppy/pTS/NT3 and compared to stimulated or unstimulated

Ppy/pTS (po0:001). (d) Release of 125I-NT3 from stimulated (grey) and

unstimulated (black) 1 cm2 Ppy/pTS/125I-NT3 polymers was measured

daily, as well as immediately after the stimulation period. A greater

amount of 125I-NT3 was detected in solutions of electrically stimulated

samples compared to unstimulated Ppy/pTS/125I-NT3. Error bars

represent standard error of the mean. Ppy refers to Ppy/pTS, ‘stim’ refers

to 1 h stimulation and Ppy/NT3 refers to Ppy/pTS/NT3. �po0:001compared to all other means. Scale bars are 100mm.

Fig. 8. Impedance of gold and Ppy-coated electrodes. Impedance values

were measured from the voltage waveform arising from a 1.5mA biphasic

current pulse (i) with 50 ms pulse width. Impedances were calculated from

the voltage waveform (inset) and Ohm’s law. Total impedance (Zt) was

calculated from the peak voltage output (Zt ¼ V t=i), access resistance

from the initial voltage drop (Ra ¼ Va=i) and polarisation impedance

from the remaining voltage drop (Zp ¼ Vp=i). There was no difference

in Zt, Ra or Zp between plain gold electrodes, Ppy/pTS-coated gold

electrodes and Ppy/pTS/NT3-coated gold electrodes when measured in

phosphate buffered saline at room temperature. Ppy refers to Ppy/pTS

and Ppy/NT3 refers to Ppy/pTS/NT3. Error bars denote the standard

error of the mean.

R.T. Richardson et al. / Biomaterials 28 (2007) 513–523520

survival of SGNs within the explant but does not directlycorrelate with the number of surviving SGNs within theexplant due to the bipolar nature of auditory neurons andoccasional branching that occurs within the explant.Counting neurites at the nearest point of exit from theexplant excludes branching effects that occur distal to theexplant, minimising further double-counting of neurites.There are image analysis techniques that measure com-bined neurite outgrowth and length [36], but neurites in thecochlea do not have to grow over large distances as they doin other applications such as spinal cord repair. Therefore,this paper focuses on quantity of SGN neurite outgrowthover neurite length.

4.2. Growth of auditory neurons on Ppy

The presence of NT3 in the culture media or a CAMcoating on each substrate (plastic, Ppy/pTS and Ppy/pTS/NT3) resulted in increased neurites extending from SGNexplants. Importantly, explants grown on Ppy/pTS/NT3yielded more neurites per explant compared to those grownon Ppy/pTS or plastic regardless of CAMs, suggesting thatthe augmented neurite outgrowth occurred via release ofNT3 from the Ppy rather than difference in surfacechemistries of the polymers. In support of this, it wasfound that explants that were not in direct contact withPpy/pTS/NT3 grew equally well as explants growing on thesurface of Ppy/pTS/NT3.

ARTICLE IN PRESSR.T. Richardson et al. / Biomaterials 28 (2007) 513–523 521

It has been shown here and previously that there wasminor diffusion of NT3 from Ppy/pTS/NT3 in the absenceof electrical stimulation [23]. Our results suggest that theNT3 diffusing out is biologically active and is conferring abenefit to neurite outgrowth. The amount of NT3 diffusingfrom unstimulated Ppy/pTS/NT3 was very low (1.15 ng/mlwhen the unstimulated release experiment was examined atday 3 and adjusted for electrode size and culture volume)compared to the 40 ng/ml added to the media in somegroups. One explanation for the increase in neuriteoutgrowth from explants grown on these polymers is aproximity effect, in which explants grown directly on asurface releasing incorporated NT3 ‘see’ more NT3 thanthe concentration measured in solution.

NT3 incorporation studies using 125I-labelled NT3predict that the 18.0–21.3mm2 Ppy/pTS/NT3-coated goldelectrodes would contain 16.8–19.9 ng NT3 based on anaverage 93.476.6 ng/cm2 NT3 incorporated for the poly-mers used in this study (n ¼ 39) [23]. SGN survival andneurite outgrowth from explants grown on Ppy/pTS/NT3without added NT3 emphasises the point that Ppy/pTS/NT3 may be used as an NT3 delivery vehicle for elicitingSGN survival and neurite outgrowth. Addition of a CAMcoating to Ppy/pTS/NT3 or NT3 to the media had anadditive effect on neurite outgrowth suggesting that neither40 ng/ml exogenous NT3 nor the amount of NT3 diffusingfrom Ppy/pTS/NT3 is producing the maximum SGNsurvival and neurite outgrowth achievable in the explantassay.

There was no statistical difference between neuriteoutgrowth from explants grown on tissue culture plasticand Ppy/pTS suggesting that Ppy/pTS is not toxic toSGNs. Furthermore, it was shown that both of theconstituents of Ppy/pTS, namely monomeric pyrrole andpTS, had no adverse effects on neurite outgrowth at thehighest concentrations used in culture. In another study,monomeric pyrrole was injected intraperitoneal andintravenously for acute and sub-acute toxicity tests inmice, rabbits and guinea pigs without any obvious adverseeffects. Additionally in this same study, 6 months ofPpy–silicone implantation in rat peripheral nerve tissueresulted in only mild inflammation [14].

4.3. Neural and cellular interactions with Ppy

When viewed under SEM, Ppy/pTS and Ppy/pTS/NT3polymers had a globular appearance supporting previousfindings of Ppy morphology [37]. We found that theglobular surface did not inhibit cell attachment or neuriteoutgrowth. Cells and neurites appeared to attach to theraised globules of Ppy, although this might be an artefactof SEM if the critical point drying caused greater cellshrinkage compared to the Ppy surface. An alternativehypothesis is that the raised globules of Ppy have adifferent charge density, attracting cell attachment to thesepoints. Neurite and cell growth across gaps of 50 mm havebeen observed with no points of attachment in between

demonstrating that cells need not follow the contours ofthe surface on which they grow [38].There is evidence suggesting that incorporation of

neurotrophins into Ppy enhances nerve growth activityin vivo around the site of implantation. Incorporation ofnerve growth factor, a neurotrophin related to NT3 instructure and function, into Ppy increased the amount ofneural tissue around the polymer following implantation inthe rat brain [13]. The results described here suggest thatPpy can be used both as an agent that can deliverneurotrophins to sites of nerve injury but also as aplatform on which regenerating nerve fibres can grow.

4.4. Electrical stimulation

The electrical stimulus used in this study was designed tosimulate the electrical stimulus of a cochlear implant.Cochlear implants use charge balanced biphasic currentpulses with shorting between phases to reduce the build-upof direct current at the electrodes [39]. Clinical pulses rangebetween 20 and 200 ms per phase and have currents between10 mA and 2.5mA [40]. The charge density used in thisstudy is at the lower end of charge densities occurring incochlear implant electrodes due to the large electrodesurface area and to enable stimulation of cells growndirectly on the electrodes. However, an improved nerve–e-lectrode interface in which SGNs grow closer to or directlyon electrodes using polymers such as those studied here,would enable cochlear electrode arrays to be operated atreduced charge densities since current spread would beminimised. This would result in reduced power require-ments and improved control over the neural response toelectrical stimulation.Explants grown on Ppy/pTS/NT3 and subjected to 1 h of

biphasic current pulse stimulation had greater neuriteoutgrowth compared to explants grown on unstimulatedPpy/pTS/NT3. In contrast, electrical stimulation of Ppy/pTS did not enhance neurite outgrowth compared tounstimulated Ppy/pTS. These results suggest that theenhanced neurite outgrowth observed from SGN explantsis due to release of NT3 from the polymer rather than theelectrical stimulus. Supporting this, the 125I-NT3 releasestudy showed enhanced NT3 release over the 1 h stimula-tion period and greater overall release over the 4 days ofincubation. Adjusting for the size of the polymers andincubation volumes in the electrical stimulation study,unstimulated Ppy/pTS/NT3 used in cultures would havereleased 1.19 ng/ml NT3 while stimulated Ppy/pTS/NT3would release 1.35 ng/ml NT3 over 4 days. The proximityeffect described earlier or a combined effect of NT3 andelectrical stimulation might account for the differencesobserved in neurite outgrowth between stimulated andunstimulated groups.Many in vitro studies with PC12 cells or dorsal root

ganglia demonstrate enhanced neurite outgrowth in thepresence of electrical stimuli [41–43]. However, these areusually constant voltage electrical stimuli that have been

ARTICLE IN PRESSR.T. Richardson et al. / Biomaterials 28 (2007) 513–523522

shown to be unsafe for use with platinum electrodes in theinner ear [44]. Biphasic current pulse electrical stimulationin deafened animals has produced mixed results withrespect to survival of SGNs. One study found thatelectrical stimulation alone was not sufficient for nervesurvival after sensorineural hearing loss in cats, requiringinstead combined treatment with neurotrophic factors [24].Others found that electrical stimulation alone preservedSGNs in deafened cats or guinea pigs [45,46]. From thesestudies, we cannot assume that electrical stimulation alonewill have a trophic effect on SGNs. Our results showed thatelectrically stimulated Ppy/pTS/NT3 releases biologicallyactive NT3 at levels sufficient to promote neurite out-growth from auditory neuron explants in vitro.

Ppy-coated electrodes would not benefit the cochlearimplant if the coating increases the electrode impedanceas this would increase the power consumption of thedevice. A coating of Ppy/pTS or Ppy/pTS/NT3 did notcause significant changes in impedance of gold electrodes,suggesting that Ppy could be used to coat cochlear implantelectrode arrays without adversely affecting their capacityto deliver electrical charge to SGNs. The gold electrodesused in this study are a suitable model for examiningchanges that occur on platinum electrodes of cochlearimplants as their electrical and bio-interactive propertiesare similar to those observed with platinum in vivo

electrodes [26]. While there are important differencesbetween cochlear implant electrodes and the electrodesused in this study (including size, material and shape), ourobservations suggest that Ppy/pTS/NT3 coatings mayimprove the performance of the cochlear implant bymaintaining SGN survival and promoting their regenera-tion after sensorineural hearing loss through the release ofNT3.

5. Conclusions

To encourage neuroprotection and nerve fibre growthtowards the cochlear implant electrodes, we propose acoating of the electrodes with an electrically conductingPpy/pTS charged with NT3. This study demonstrated thata Ppy/pTS/NT3 coating of electrodes provides a biocom-patible substrate for nerve growth that acts as a store ofneurotrophins and a controlled release device. Theneurotrophins retain their biological activity throughoutthe entire process and are released in quantities thatpromote auditory neuron survival and neurite outgrowth.Electrical stimulation with cochlear implant stimulationparameters accelerates the release of NT3 from thepolymer and hence enhances neurite outgrowth further.

Given the small fluid volume of the cochlea, the amountof NT3 released from polymer-coated electrodes demon-strated here is sufficient for auditory neuron survivalin vivo. In addition, the low impedance of the polymercoating does not diminish the electrical function ofelectrodes facilitating the likelihood that a cochlear implantcoated with Ppy/pTS/NT3 will function normally and

provide a mechanism to release NT3 for SGN survivalafter sensorineural hearing loss.

Acknowledgements

This work was generously funded by the Friends of FlintHill Foundation, the ARC Centre of Excellence forElectromaterials Science and the Bionics Program of theARC Centre. The authors would like to thank RodneyMillard for engineering the connectors for culture slidesand for valuable input into discussions on electricalstimulation. Thanks also go to Dr. James Fallon foradvice on statistical analysis and to Bryony Coleman andBrianna Flynn for proof reading. The Zeiss Axioplanmicroscope was purchased with funds from the NIDCD(N01-DC-3-1005). The authors are grateful for the use ofthe biological cabinets in the teaching laboratories of theSchool of Biological Sciences, University of Wollongong,for sterile polypyrrole polymerisations.

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