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Biomaterials 31 (2010) 886–893

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Biomaterials

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Cytotoxicity of implantable microelectrode arrays produced bylaser micromachining

Rylie A. Green a,*, Juan S. Ordonez b, Martin Schuettler b, Laura A. Poole-Warren a, Nigel H. Lovell a,Gregg J. Suaning a

a Australian Vision Prosthesis Group, Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW, 2052, Australiab Laboratory for Biomedical Microtechnology, Department of Microsystems Engineering, University of Freiburg, Germany

a r t i c l e i n f o

Article history:Received 28 July 2009Accepted 29 September 2009Available online 15 October 2009

Keywords:Laser micromachiningMicroelectrodesCytotoxicityHigh-density arrayNeuroprostheses

* Corresponding author.E-mail address: r.green@unsw.edu.au (R.A. Green)

0142-9612/$ – see front matter Crown Copyright � 2doi:10.1016/j.biomaterials.2009.09.099

a b s t r a c t

Implantable high-density microelectrode arrays have been successfully fabricated using laser micro-machining of conventional implant materials, polydimethylsiloxane (PDMS) and platinum (Pt) foil. Thisstudy investigates the impact of modifying PDMS and Pt with high power laser beams and the possibletoxicity of by-products that may remain on the implantable device. Materials were characterised bothchemically and biologically through x-ray photoelectron spectroscopy (XPS), cell growth inhibitionassays and a direct contact cell proliferation assay. It was found that laser micromachining producesoxides of silicon and platinum on the PDMS and Pt respectively. While the chemical properties ofmaterials were altered, there was negligible change in the biological response to either extracts or cellgrowth directly on the composite electrode array.

Crown Copyright � 2009 Published by Elsevier Ltd. All rights reserved.

1. Introduction

A complex issue in the field of neuroprostheses is the developmentof high-density microelectrode arrays to evoke or record neuralactivity. It is thought that the provision of a higher density array ofelectrodes in electrical neural stimulation will allow a greater numberof discrete neurons or groups of neurons to be activated and corre-spondingly result in increased localisation and control of the desiredbiological response. For example, one hypothesis describes thatincreasing the number of cochlear implant stimulation sites will leadto improvements in patient speech perception and music apprecia-tion [1,2]. While the minimum size of electrodes and the optimalelectrode number is a subject of intense research, manufacturinglimitations have hindered progress in the development of high-density microarrays [2,3]. Recently, the Australian Vision ProsthesisGroup (AVPG, University of New South Wales, Australia) in collabo-ration with the Laboratory for Biomedical Microtechnology at theDepartment of Microsystems Engineering (IMTEK-BMT, Freiburg,Germany) have developed a method for the fabrication of complexhigh-density microelectrode arrays by employing laser micro-machining technology [4]. These flexible constructs were designed forvision prosthesis [5] and cortical recording applications [6], but theversatility and ease of processing methods give this technology the

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potential to be applied across a wider scope of implant and bioma-terials applications.

The high-density microelectrode arrays are comprised of mate-rials similar to those previously approved by regulatory bodiesthroughout the world for use in other medical applications such ascochlear implants [7], non-cardiac pacemakers [8] and breastimplants [9]. Despite the known biological response to these mate-rials in the listed applications, the effect of specific processing tech-niques required to manufacture these arrays has not beenestablished. Specifically, the impact on biological systems of possibleby-products formed during laser micromachining multi-layeredconstructs of polydimethylsiloxane (PDMS) and platinum (Pt) foilhas not been assessed. Conventional bioelectrodes such as the Neu-ronexus and Cyberkinetics cortical recording electrode arrays andthose used in cochlear implants are fabricated by way of micro-machining, microwire technology and/or photolithography tech-niques [10,11].

Micromachining is typically used for producing rigid electrodesbased on silicon wafer platforms [12], but is not ideal for fabricationof flexible arrays, required for vision prosthesis devices and corticalimplants with low mechanical impact. Microwire technology islabour intensive, requiring some manual input during fabrication,and also requires a considerable volume of wire to produce a high-density of contact points. While photolithography has allowed thedevelopment of flexible arrays with a wide variety of designs, thismethod is not reproducible and uncertainty usually remains aboutthe exact shape of the resulting electrode contacts [13]. The method

rights reserved.

R.A. Green et al. / Biomaterials 31 (2010) 886–893 887

of fabrication designed by Schuettler et al. addresses these limita-tions, being simple, repeatable and applicable to a wide variety ofelectrode designs, shapes and densities [5]. Laser micromachiningusing an Nd:YAG laser at 1064 nm wavelength allows fabrication ofelectrode contacts through composite materials [5], however theeffect of laser application to conventional implant materials and thetemporary involvement of non-implantable materials employedduring electrode manufacture need to be examined prior to clinicalstudies.

Application of laser fabrication techniques to PDMS is reported inliterature for the modification of surfaces to achieve controlled cellinteractions [14–16]. In these reports PDMS and PDMS co-polymershave been exposed to low-power laser beams which inducemorphological changes in the PDMS surface. The surface chemistryof these materials remains unchanged, however, the topographiesare altered to control cell adherence and growth characteristics. Ingeneral it was observed that fibroblastic growth was minimised onlaser roughened PDMS due to an increase in hydrophobicity,induced by the modified surface morphology [15,16].

Laser roughening of metals has also been explored for improvingthe charge transfer properties of implant electrodes [16,17]. It hasbeen reported by a number of groups that modifications whichincrease the electrode surface area across a fixed geometric base,result in better charge transfer due to the ability to use higher chargedensities within the safety limits of the electrode material [2,17–21].Additionally, laser microstructuring of Pt electrodes has beeninvestigated for directing the growth of neural cells at the electrodeinterface [16]. It has been shown that fibroblastic cells and neuronalcells respond to specific surface topographies, and the control of thisproperty during fabrication will influence the type of cell growthseen at the electrode interface [16,18]. A desirable surface will beoptimised for neural cell interactions with the Pt stimulating portion,while resisting the formation of scar tissue through providing a non-conducive environment for fibroblastic cell attachment on the PDMS.

Most previous reports on laser modifications of both PDMS and Ptfocus on the use of controlled low-power beams for surfacedisruption. Regardless of the application, these studies have aimed tocreate materials with increased surface area [16–18]. There is a lackof literature reports on the chemical and biological impact of struc-turing PDMS and Pt using high-power lasers. The aim of this studywas to determine the chemical composition and cytotoxicity of theby-products produced in laser micromachining processes and theirpotential effect on biological systems compared to conventionallyprocessed controls. Specifically, the chemical composition of affectedareas of the microelectrode array were examined by X-ray photo-electron spectroscopy (XPS) and two in vitro cell assays were used todetermine the possible toxic effects of material modificationsresulting from fabrication processes. An L929 direct contact

Fig. 1. 29-channel cortical microelectrode array fabricated by laser-structuring of Ptfoil and PDMS.

proliferation study and a cell growth inhibition assay were used toassess the effect of material processing on biological systems.

2. Materials and methods

Microelectrode arrays such as the array shown in Fig. 1 have been successfullyfabricated using the technology presented by both Schuettler et al. and Suaning et al.[4–6]. This method of laser micromachining follows a vector path created withcomputer-aided design (CAD) software. The laser is a diode pumped, Q-switchmanaged, pulsed Nd:YAG laser (Class 4) with constant wave (CW) power of 7 watts(DPL Genesis Marker, CAB GmbH, Karlsruhe, Germany). The optical power level ofthe beam and its effects on the processed material can be adjusted throughparameters such as the percentage of the CW power, scanning velocity along thevector path, Q-switch frequency and Q-switch opening time. Variation of theseparameters is used to achieve the desired results on the targeted materials.

2.1. Fabrication of electrode arrays

The processing method for the test samples in this study was based on thepreviously developed electrode fabrication method [4–6]. Briefly, the process wasconducted as shown in Fig. 2 and involved initially depositing a release layer such asagar (BBL Agar, Grade A, BD Biosciences, San Jose, CA, USA) or laminating self-adhesive silicone tape (No. 4124, Tesa� AG, Hamburg, Germany) onto a standard glassmicroscope slide (Fig. 2-1). This was followed by spin coating the PDMS (MED-1000,NuSil, Carpinteria, CA, USA) diluted in N-heptane on to the slide to approximately25mm in thickness (Fig. 2-2) and �C for 15 min. The MED-1000 is a restricted variantof PDMS approved for implantation for up to 29 days [19]. This one-part, self-levellingPDMS was found to be superior in producing uniform spin-coated layers whencompared to a variety of two-part alternatives [20]. The chemical composition andphysical properties of MED-1000 are identical to the more expensive unrestrictedMED-2000 PDMS (approved for chronic implantation over 29 days) [21], whichwould be employed in the clinical device. For this stage of development and testingprior to long-term implantation the restricted variant will provide identical results ata much lower cost. The release layer counteracted the high adhesion characteristicsof PDMS on glass, which in direct contact, prevents the removal of the finishedflexible electrode from the fabrication substrate. A 12.5mm thin foil of 99.95% plat-inum (Pt) (Goodfellow, Huntingdon, UK) was laminated onto the PDMS surface and

Fig. 2. Cross-sectional schematic of stepwise electrode fabrication by laser micro-machining; 1. Placement of release layer; 2. Spin coat PDMS; 3. Placement of Pt foil;4. Laser patterning of Pt electrode constructs; 5. Manual removal of Pt excess; 6. Spincoat of PDMS; 7. Laser mediated crystallisation of PDMS; 8. Laser pulse removal ofcrystallised PDMS; 9. Laser cutting of PDMS border; and 10. Release of entire electrodearray from support substrate.

Table 1Samples assessed for impact of various laser micromachining techniques on implantmaterials.

Sample Material Laser action Underlying layer

1 PDMS (control) – –2 PDMS Cutting Agar3 PDMS Cutting Tesa�

4 PDMS Crystallisation Platinum5 Pt (control) – –6 Pt Ablation PDMS

R.A. Green et al. / Biomaterials 31 (2010) 886–893888

the structures were cut into the foil by laser ablating the Pt along a vector definedpath (Fig. 2-4). The excess Pt foil is manually removed from the slide (Fig. 2-5) anda second layer of PDMS was spin-coated and cured at 60 �C for 20 min, to embed thelaser-machined structures (Fig. 2-6). Where the PDMS required removal to exposeunderlying metals, such as opening the electrode contacts (Fig. 2-7), combustion ofthe PDMS was laser induced to locally change the polymeric microstructure intoa crystalline configuration. These crystals were subsequently removed with low-power laser pulses (Fig. 2-8) without adversely affecting the underlying metallicstructures. The contour of the electrode was defined by laser-cutting the PDMS downto the release layer (Fig. 2-9). Finally, the finished electrode array was mechanicallyremoved from the carrier substrate and cleaned.

2.2. Fabrication of extraction test samples

Tailored test samples were fabricated to examine the evolution of by-productsproduced during the laser fabrication steps described above. Sample schematicsrepresentative of the specific modifications of interest are depicted in Fig. 3. Thesesamples were designed to investigate the three variations in laser application,including crystallisation of PDMS over Pt-foil for electrode exposure (Fig. 3A),cutting of the PDMS down to the underlying release layer (Fig. 3B) and ablation of Pt-foil over a PDMS layer (Fig. 3C). The two release layer variants, Tesa� tape and agar,were investigated to determine the effect of the possible different by-productsarising from these underlying materials which may be altered during laser cutting ofthe microelectrode array contour.

In accordance with ISO 10993 an exposed area of 15 cm2 was required for eachmaterial sample used in the cell growth inhibition assay [25]. In this study a worst-case scenario was used such that each material composite machined by the laser wasassessed rather than the more conventional assessment of the final implantableelectrode array. For example, laser cutting of the external border of the flexible arrayis used to release the electrode from the surrounding excess PDMS and as such, thetoxicity of by-products evolved from this process was assessed using a samplewholly composed of laser-cut PDMS, as depicted in Fig. 3A. The structured area ofeach sample had external dimensions of 60 mm� 35 mm, however, the depth of thecut and the increased surface roughness at the edge of ablated segments substan-tially increases the surface area of the material under test. The final area cannot beaccurately determined, but the samples were designed to achieve geometric area of15 cm2 (considering a surface roughness of zero) for all constructs which required

Fig. 3. Cross-sectional schematic of test sample composition, A. Crystallisation ofPDMS, B. Cutting of PDMS down to the release layer, and C. Ablation of the Pt.

testing, as listed in Table 1. Samples produced on agar release layers, including thecontrol samples, were released by boiling in water for 20 min. Samples produced onTesa� tape were lifted at the edges and then a small amount of ethanol was placed inthe gap between the sample and slide.

2.3. Scanning electron microscopy

Scanning electron microscopy (SEM) was used to visualize the material surfacemorphology following laser micromachining processes. Electrode constructs werecoated with an 8–12 nm layer of gold using an EmiTech K550 high resolution coater(EM Technologies Ltd., Ashford, UK). The samples were placed in a JEOL JSM-5800SEM (JEOL Ltd., Tokyo, Japan) under high vacuum with an accelerating potential of5 kV. Images of the electrode constructs were captured at 150� magnification. Thelaser affected regions were visualized at higher magnification varying between1000� and 3500�, and compared to unaffected regions beyond the laser structuring.

2.4. X-ray photoelectron spectroscopy

XPS was carried out (Kratos XSAM800 XPS, Japan) and peaks were analysed todetermine the elemental constitution of the electrode materials following lasermicromachining. The samples listed in Table 1, were fabricated as previously dis-cussed but with an area of approximately 25 mm2 to facilitate placement in the XPSchuck. Control samples consisting of the untreated Pt and PDMS were used todemonstrate the relative changes in chemical composition.

Curve fitting was performed (Eclipse Datasystem V2.1, USA) and broad peakswith the same width were used in preliminary analysis. Where curves were found tocontain more than one chemical state of a given species, the PeakFit function wasused to elicit the different chemical states through the use of an additional peakwidth resulting in the most accurate fit available. The intensity ratio of the doubletwas maintained at 2:1 and the energy separation was maintained at 1.18 eV.

2.5. Cell growth inhibition

The samples listed in Table 1 were extracted at 70 �C for 24 h in saline(Baxter’s 0.9% NaCl, Baxter Healthcare Pty. Ltd., NSW, Australia). Controls of latex(positive control), ethanol (4% EtOH, 5%EtOH and 7.5% EtOH assay controls) andduplicate extraction controls (glass vials) were included. A fibroblast monolayer(L929 mouse fibroblasts, ATCC CCL-1) was prepared by seeding at 105 cells/mL incomplete media (Eagle’s minimum essential media (EMEM, Sigma–Aldrich,Australia) with 10 vol% fetal bovine serum (FBS, JRH Biosciences, Sigma–Aldrich,Australia)) supplemented with 2% penicillin/streptomycin (P/S, CSL Biosciences,Edwardstown, SA, Australia) and incubating for 24 h at 37 �C and 5% CO2. Afterremoval of media and washing of the cell layer, extraction fluid and control fluidswere applied to the cell monolayers and incubated for 48 h. Null samples werenot exposed to extraction fluid, but were replenished with fresh complete mediaand incubated under identical conditions.

After 48 h, monolayers were washed with Dulbecco’s phosphate buffered salinewithout calcium and magnesium (DPBS, Sigma–Aldrich, Australia), trypsinised(0.12% trypsin, 0.02% ethylene diamine tetra-acetic acid (EDTA), 0.04% glucose, JRHBiosciences, Sigma–Aldrich, Australia), and assessed by Vi-Cell counter (BeckmanCoulter, Australasian Analytical Systems, QLD, Australia) to determine viable cellnumbers. Results for each study were expressed as a ratio of the viable cell numberscounted for the test samples versus the untreated control materials. As such resultsfor the ablated Pt were given as a ratio to the untreated Pt and all other samples,which were laser modifications of the PDMS, were given as a ratio to the untreatedPDMS. In each assay three replicate extraction samples were analysed for each testmaterial and the entire experiment was repeated three times to achieve statisticalvalidity of N¼ 3. An analysis of variance (ANOVA) with a Tukey test was performedusing Minitab (Minitab Pty Ltd, Sydney, Australia) to assess any statistical differencesbetween the test samples.

2.6. Direct contact proliferation assay

This direct contact study of cell growth utilises L929 fibroblasts cultured directlyon the laser machined microelectrode arrays fabricated as described in Section 2.1.Untreated component samples of Pt foil and PDMS were used as process controls,with tissue culture plastic (TCP) well plates (BD Biosciences) and latex providing thepositive and negative controls, respectively. The electrode array was produced suchthat Flexiperm silicone gasket wells (Greiner BioOne, Chicago, Il, USA) could beclamped over the system to contain medium during the assay period. The PDMS andPt samples were mounted on the slide during fabrication of the electrode andsimilarly contained by the gasket well system. Disinfection was performed byexposing the wells to ultra violet (UV) radiation for 2 h.

L929s were plated on the various samples and three wells of the 96-well TCP at104 cells/cm2. Two sample sets were studied, with the first set analysed at 4 h andthe second cultured for 48 h in complete medium (Dulbecco’s minimum essentialmedia (DMEM, Sigma–Aldrich)þ 10 vol% FBS) supplemented with 2% penicillin/streptomycin at 37 �C, 5% CO2.

0100200300400500600700800900Bond Energy (eV)

Laser ablated PDMS on platinum

Laser cut PDMS on agar

Unprocessed PDMS

O1s

C1s Si2s Si2p

Laser cut PDMS on Tesa

Fig. 5. Sample XPS of PDMS showing chemical modifications resulting from laserfabrication processes.

Fig. 4. Sample SEM of electrodes and component materials following laser micromachining. A. Sample electrode construct at 150� magnification; B. Pt foil after laser opening ofelectrode contact; C. Untreated Pt foil; D. Crystallised PDMS before removal by low-power laser pulses; E. PDMS after removal of excess crystallised material; F. Laser cut PDMS(border region of electrode array with top surface visible); and G. PDMS from unaffected region of electrode array. Samples B–G taken at 1200� magnification.

0100200300400500600700800900Bond Energy (eV)

Laser ablated Pt on PDMS

Unprocessed Pt on PDMS

O1s

C

1

s

S

i

2

s

S

i

2

p

P

t

4

d

3

P

t

4

d

5

P

t

4

f

Fig. 6. Sample XPS of Pt showing chemical modifications resulting from laser ablation.

R.A. Green et al. / Biomaterials 31 (2010) 886–893 889

At 4 h and 48 h the L929 cells were live stained using Calcein-AM (1 mg/mL inDPBS incubated for 20 min) and Hoechst 3342 (Invitrogen, Vic, Australia) nuclearstain (0.5 mg/mL in DPBS incubated for 10 min). After the stain uptake by living cells,assays were analysed through fluorescent micrographs (Carl Zeiss Vision, NSW,Australia) and quantified by employing software package Image J (National Instituteof Health, USA) to count the viable cells in each image. Increase in cell numbers wasassessed by calculating the ratio of adherent cell numbers at 48 h to that at 4 h andexpressing as a percentage. Three individual samples were analysed for eachmaterial type at both time points and the entire experiment was repeated threetimes to give N¼ 3. An ANOVA with a Tukey test was performed using Minitab(Minitab Pty Ltd, Sydney, Australia) to assess any statistical differences between thetest samples. The cell proliferation for each material was plotted with mean stan-dard error.

3. Results

3.1. SEM

Surface morphology of the electrode component materialssubstantially changed following laser micromachining as shown inFig. 4. A roughened region of PMDS surrounded each exposedelectrode contact for a width of approximately 50 mm and thecentral Pt region, magnified in Fig. 4B, is also substantially altered

Table 2Elemental composition of PDMS surface after laser modification on varioussubstrates.

Elemental composition (%)

Element O1s O1s C1s C1s C1s Si2p Si2pPeak centre 534 532 288 286.6 285 104 102

Untreated PDMS 23.8 48.6 27.6Laser cut PDMS on agar 0.9 26.7 1.4 2.2 41.5 3.5 24.7Laser cut PDMS on Tesa� 30.6 39.8 4.7 24.9Laser ablated PDMS on Pt 55.9 0.5 7.5 36.1

Table 3Elemental composition determined by XPS of Pt samples following laser ablation.

Elemental composition (%)

Element O1s O1s C1s C1s C1s C1s Si2p Pt4f7 Pt4f7 Pt4f7Peak centre 532 531 289 288 286.6 285 102 75 73 71.8

Untreated Pt 24.5 2.8 2.5 3.2 34.5 2.2 18.1 – 3 9.2Laser ablated Pt on

PDMS28.4 6.6 1.3 1.2 1.6 34.6 14.6 4.8 4.7 2.2

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

Pt -Abla

ted

PDMS -Cut

on ag

ar

PDMS -Cut

on Tes

a

PDMS -Crys

tallise

d

Ra

tio

o

f v

ia

ble

c

ell n

um

be

rs

(T

es

t s

am

ple

/U

np

ro

ce

ss

ed

c

on

tro

l)

Study 1Study 2Study 3Average

∗

Fig. 7. Ratio of viable cell numbers to untreated controls at 48 h following exposure totest sample leachants. Asterisk indicates no significant difference (NSD) as determinedby ANOVA; (N¼ 3).

R.A. Green et al. / Biomaterials 31 (2010) 886–893890

with a considerably more textured surface than unaffected Pt(Fig. 4C). The crystallised PDMS is shown in Fig. 4D, but it is notedthat this material is predominantly removed by low-power laserpulsing to reveal a border region with a much smoother surfacetopography, depicted in Fig. 4E. The wall region of the electrodeconstruct is pictured in Fig. 4F where the PDMS is laser-cut down tothe release layer. The cut PDMS appears to be rougher than theuntreated PDMS pictured in Fig. 4 G, but significantly less affectedthan the crystallised PDMS.

Fig. 8. Sample fluorescent micrographs of L929s stained with Calcein-AM (green) and Hoemicromachined PDMS insulated Pt electrode array.

3.2. XPS

Chemical composition of the samples was determined by XPS toproduce spectra with peak intensity representative of the relativepresence of each element. Fig. 5 shows the various spectra obtainedfor PDMS modifications and Fig. 6 shows the Pt spectra.

Curve fits were used to determine the relative peak size of eachelement and the percentage contribution of each element wasrecorded in Tables 2 and 3 for PDMS and Pt modifications respec-tively. It was found that laser modifications to the PDMS resulted inincreased amount of oxygen detected in the XPS. When the PDMSwas cut and the agar release layer exposed, additional oxygen,carbon and silicon peaks were detected. When the Tesa� releaselayer was similarly exposed only an additional silicon peak wasdetected. Laser crystallisation of PDMS on Pt resulted in a notableincrease in both oxygen and silicon, with a correspondingly largedrop in carbon.

The most significant change in the Pt spectra following Nd:YAGlaser ablation was in the dominant carbon peak, which shifted fromC1s with a peak centre at 286.6 eV to C1s with a peak centre at285 eV. This corresponds to a chemical shift in contaminatingsurface material, since carbon is not present in the commerciallypurchased Pt.

3.3. Cell growth inhibition

Cell counts performed on a Vi-Cell counter were used to plot theratio of viable cells for the test samples against the untreatedcontrols in Fig. 7. All process controls including the null, latex andethanol titrations (not shown in plot), performed as expected andconfirmed the validity of each assay. Test samples exhibited nosignificant difference to the untreated control materials. No materialleachant had a significant impact on cell growth following laserablation, crystallisation or cutting. While the sample produced fromcut PDMS on the agar release layer did have a lower average cell

chst-3342 (blue) at 48 h post-plating on A. TCP (control); B. Pt; C. PDMS; and D. Laser

Fig. 9. Growth of L929s in direct contact with electrode component materials and the laser fabricated microelectrode array. Values given are the percentage increase in cell numberbetween t¼ 4 h and t¼ 48 h. Error bars represent SEM (N¼ 3). Asterisks indicate NSD as determined by ANOVA.

R.A. Green et al. / Biomaterials 31 (2010) 886–893 891

number than that recorded for the Tesa� tape variant, the differencewas not statistically significant.

3.4. Direct contact proliferation assay

Sample images in Fig. 8 show that cell spreading was optimal onthe positive control, TCP, but cells were more rounded on both theelectrode array and the component samples. Rounded cells areindicative of poorer interactions between the cells and substratematerials. It is also noted that on the array (Fig. 8D) most cells areconfined to the Pt portion of the composite electrode, with few cellsshowing preference for the PDMS portion, despite the PDMScomprising more than 50% of the total available area. Cells were notdetected on the latex negative controls (image not shown).

Cell proliferation plotted in Fig. 9 demonstrates that growthoccurred on all materials except the latex negative control, wherecell death was prevalent. While none of the electrode materialsperformed as well as the TCP positive control, PDMS and Pt, whichare widely accepted as being biostable materials, had proliferationrates that were not significantly different to the laser micro-machined electrode array constructed from these materials.

4. Discussion

Laser micromachining technology has been reported asa method for producing complex high-density electrode arraysutilising materials commonly used in regulatory approved, chron-ically implanted devices [4,5]. SEM demonstrated that importantmorphology changes remain at the implant surface following lasermicromachining. An XPS chemical analysis and two cell basedassays were used to examine material changes resulting from theseprocessing techniques intended for the fabrication of implantablemicroelectrodes. Cell based cytotoxicity assays are a widelyaccepted preliminary step toward ensuring the biocompatibility ofa medical device’s material composition. It is important to note thatin these studies the material samples were provided in substan-tially higher quantities than would normally be present in animplant. For example, in a high density 98-electrode array intendedfor use in a vision prosthesis a nominal 3 mm2 of PDMS would becrystallised, which is 500 times less surface area than the areatested in the cell growth inhibition assay.

XPS analysis showed a shift in the chemical constitution of allmaterials following application of laser processing, with mostmaterials experiencing increased formation of oxides. All PDMSvariants showed an increase in the presence of both silicon and

oxygen, most likely owing to the presence of SiO2 produced fromexposure to the high-power laser radiation in the presence ofoxygen. The PDMS on the agar release layer also had additionalpeaks indicative of the presence of organic matter at the materialsurface. This is not unexpected as the agar consists primarily of thelinear peptide agarose, with the chemical structure{C12H14O5(OH)4}n. In comparison, the PDMS on Tesa� tape showedonly a shift in peaks consistent with the formation of SiO2 followingevaporation. While the Tesa� tape is silicone based, the exactchemical composition is unknown and hence potential by-productsmay be masked by the PDMS spectra. The dominant by-productdetected at the PDMS surface, SiO2 has an LD50 (lethal dose for 50%of the test population) of 3160 mg/kg (administered orally in rats)[26] compared to PDMS with an LD50 of 40,000 mg/kg [26]. Whilethis indicates that an increase in toxicity may occur as a result ofhigh-power laser processing of implant materials, both cell studiesshow a negligible change in cell growth characteristics whenexposed to the laser modified PDMS. It is proposed that this is due toadequate removal of the crystallised PDMS by the low-power laserpulsing. The SEM shows that large, nodular particles of modifiedPDMS are present at the electrode surface following crystallisation(Fig. 4D), but the low-power laser pulsing removes the majority ofthis debris (Fig. 4E), minimising the exposure of tissue to SiO2.

Similarly, the XPS of the Pt demonstrates that oxides of platinumare formed at the material surface during laser ablation. Specifically,the Pt 4f7 peak shifts to a higher energy level consistent with theformation of PtO2. In this instance the Pt has an LD50 of 9100 mg/kgcompared to by-product, PtO2, with an LD50 of 8000 mg/kg [27]. Itis unlikely that laser micromachining techniques which produceoxides of Pt will impart any increased toxicity to the implantsystem. Both the cell growth inhibition assay and the direct contactproliferation assay support these findings. In fact, metal oxidesare predominantly less reactive than the metal itself whensurface expressed due to passivation which minimises loss ofpotentially toxic ions, and therefore may be more supportive of cellgrowth [28].

The direct contact proliferation assay determined the rate ofL929 growth on untreated medical grade PDMS, Pt foil and thecomposite laser micromachined electrode array over a 48 h period.All values were normalised to the TCP negative control where therate of L929 proliferation was optimal. Growth rates on theuntreated PDMS and Pt compared to the laser fabricated micro-electrode array did not show significant difference. Combined withthe results from the extraction study, this confirms that any by-products formed during the fabrication of the electrode are either

R.A. Green et al. / Biomaterials 31 (2010) 886–893892

non toxic, or alternately, not present in quantities sufficient to effectthe surrounding biological environment.

Studies by Reuter et al. which assessed L929 proliferation oncochlear implants with femtosecond laser microstructuring, indi-cated similar results for their laser processing techniques [18]. Inthis application, two types of silicon and Pt were assessed followinglaser mediated surface roughening. It was reported that cell growthoccurred on all materials but was lower on the PDMS than on Pt[18]. The report by Reuter et al. concurs with this study wherefibroblastic cells preferentially adhered to the Pt portion of theelectrode construct with few cells (<25%) attaching to the PDMS,despite comprising 52% of the available cell growth area. This is notunexpected as several groups in the literature have noted theincreased hydrophobic properties of PDMS following laser modi-fications, which serves to increase surface roughness [14–16,18].The hydrophobic PDMS resists the attachment of proteins presentin the media and hence the PDMS portion of the electrode constructis less interactive with the cells compared to the Pt.

The surface morphology of the Pt electrode contacts followinglaser removal of the insulating PDMS is hypothesised to benefitimplant interactions at the neural interface. A number of materialprocessing techniques which increase the available surface areaacross a fixed geometric base have been reported to improve theelectrical properties of bioelectrodes [17,23,24,29–31]. The PDMSremoval process used in this study creates a rough topography onthe underlying Pt which is known to increase the safe limit of chargetransfer [17,22]. However, it is also known that a roughened elec-trode surface is conducive to neural cell attachment [23,24,32,33]and hence the surface morphology depicted in the electron micro-graphs (Fig. 4) is likely to be beneficial in creating a more interactiveelectrode surface than the smooth Pt produced from conventionalmicromachining techniques. Combined with the increased hydro-phobicity of modified PDMS, the surface properties of laser micro-machined electrode arrays can be tailored for specific cellinteractions. An optimal surface topography can potentially bedesigned to promote preferential attachment of neural cells to theinterfacing electrodes while preventing neural cell growth fromspanning the PDMS across multiple active sites, which can result inundesirable cross talk at the neural interface.

The laser structuring of microelectrodes, fabricated from Pt andPDMS, was first reported by Mortimer [34,35]. This researchincluded the chronic implantation of a 12-electrode cuff configu-ration around the sciatic nerve of nine cats. No significant tissuedamage was detected following 9 months of implantation [35],however, it is important to note that material composition was notthe focus of this trial. Electrodes were analysed only with respect tofunctional electrical performance and neural health within thestimulated tissue region. Although no obvious histological changeswere reported, only a minimal area of laser modified material wasexposed to the neural tissue, owing to the low number of contactsites. This study utilised PDMS variants with different chemicalcomposition and much less laser mediated microstructuring thanthat presented by Suaning et al. and Schuettler et al. [4,5], howeverit does provide positive indications for the potential in vivoperformance of high-density laser micromachined electrode arrays.

Laser micromachining techniques cause changes in thecomponent material surface morphology and chemistry, but itscontribution to overall cell behaviour is negligible. Although thefabrication process used to produce the AVPG and IMTEK-BMTelectrodes does not impact on L929 cell growth, it is evident thatmaterial surface properties will play an integral role in electrode invivo performance. Future studies will look at the impact of surfaceproperties on neural cell, glial cell and associated support tissueinteractions, and the effect of implant stimulation regimes on thematerial chemistry for chronic implantation.

5. Conclusions

While laser micromachining techniques used to fabricate high-density microelectrode arrays cause morphological and chemicalchanges at the material surface, most by-products are oxides of thecomponent materials. Cytotoxicity assays demonstrate that thesechemical changes do not impart toxic leachables, even in therigorous scenario employed here where considerably more affectedmaterial than would normally be present in an implant (around twoorders of magnitude) was tested. The laser based fabricationapproach causes negligible change in the biological response to thesematerials, assessed across cell morphology, survival and prolifera-tion. These studies suggest that laser micromachining techniques forproducing high-density microelectrodes result in viable and safestructures for long-term implants, with beneficial topographies forcell interactions in neural prosthetic applications.

Appendix

Figure with essential color discrimination. Fig. 8 of this articlemay be difficult to interpret in black and white. The full colourimages can be found in the on-line version, at doi:10.1016/j.biomaterials.2009.09.099.

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