Radiation Physics and Chemistry 80 (2011) 219–229

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Radiation Physics and Chemistry

0969-80

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F2 excimer laser (157 nm) radiation modification and surface ablation ofPHEMA hydrogels and the effects on bioactivity: Surface attachment andproliferation of human corneal epithelial cells

Zainuddin a,b,c,n, Traian V. Chirila a,b,d,e, Zeke Barnard a, Gregory S. Watson f, Chiong Toh c,e,Idriss Blakey c,e, Andrew K. Whittaker a,c,e, David J.T. Hill g

a Queensland Eye Institute, 41 Annerley Road, South Brisbane, Queensland 4101, Australiab The University of Queensland, School of Medicine, Herston, Queensland 4006, Australiac The University of Queensland, Centre for Advanced Imaging, St. Lucia, Queensland 4072, Australiad Queensland University of Technology, School of Physical & Chemical Sciences, Brisbane, Queensland 4001, Australiae The University of Queensland, Australian Institute for Bioengineering and Nanotechnology, St. Lucia, Queensland 4072, Australiaf James Cook University, School of Pharmacy & Molecular Sciences, Townsville, Queensland 4811, Australiag The University of Queensland, School of Chemistry and Molecular Biosciences, St. Lucia, Queensland 4072, Australia

a r t i c l e i n f o

Keywords:

Excimer laser

PHEMA hydrogel

Role of water

Surface chemistry

Surface topography

Cell attachment

6X/$ - see front matter & 2010 Elsevier Ltd. A

016/j.radphyschem.2010.07.036

esponding author at: Queensland Eye Institu

e, Queensland 4101, Australia. Tel.: +61 7 30

1 7 3010 3390.

ail addresses: z.zainuddin@uq.edu.au, z.zainu

a b s t r a c t

Physical and chemical changes at the surface of poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels

modified by ablation with an F2 excimer laser were investigated experimentally. An important

observation was that only the outer exposed surface layers of the hydrogel were affected by the

exposure to 157 nm radiation. The effect of the surface changes on the tendency of cells to adhere to the

PHEMA was also investigated. A 0.5 cm2 area of the hydrogel surfaces was exposed to laser irradiation

at 157 nm to fluences of 0.8 and 4 J cm�2. The changes in surface topography were analysed by light

microscopy and atomic force microscopy, while the surface chemistry was characterized by attenuated

total reflection infrared and X-ray photoelectron spectroscopies. Cell-interfacial interactions were

examined based on the proliferation of human corneal limbal epithelial (HLE) cells cultured on the

laser-modified hydrogels, and on the unexposed hydrogels and tissue culture plastic for comparison.

It was observed that the surface topography of laser-exposed hydrogels showed rippled patterns with a

surface roughness increasing at the higher exposure dose. The changes in surface chemistry were

affected not only by an indirect effect of hydrogen and hydroxyl radicals, formed by water photolysis,

on the PHEMA, but also by the direct action of laser radiation on PHEMA if the surface layers of the gel

become depleted of water. The laser treatment led to a change in the surface characteristics, with a

lower concentration of ester side-chains and the formation of new oxygenated species at the surface.

The surface also became more hydrophobic. Most importantly, the surface chemistry and the newly

created surface topographical features were able to improve the attachment, spreading and growth of

HLE cells.

& 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels maybe regarded as the prototype of biomedical hydrogels. They haveproven biocompatibility in corneal tissue, are optically clear andare not susceptible to biodegradation. Hence, they are widelyapplied in ophthalmic medicine, such as in soft contact lenses andintraocular lenses (Ruben, 1978; Barrett et al., 1986; Menapaceet al., 1989; Singh and Agrawal, 1992). In the porous forms,

ll rights reserved.

te, 41 Annerley Road, South

10 3381;

ddin@qei.org.au (Zainuddin).

PHEMA hydrogels are used as a scaffold in an artificial cornea(AlphaCorTM) (Chirila et al., 1994, 1998; Crawford et al., 2002;Hicks et al., 2003) and in an orbital implant (AlphaSphereTM)(Hicks et al., 1999a, 1999b), both marketed by AdditionTechnology Inc. (des Plaines, IL, USA). They have also beeninvestigated as a potential matrix for neural regeneration (Plantet al., 1995; Dalton et al., 2002; Flynn et al., 2003; Tsai et al.,2006). Although PHEMA has proved suitable for such applications,further extension of its biomedical applicability, particularly as asubstrate for cell attachment, spreading and growth, is hamperedby poor surface cell-adhesive properties (Ratner et al., 1975;Minett et al., 1984; Horbett et al., 1985; Lydon et al., 1985).Therefore, in line with our current research efforts to search for asuitable substrate for reconstruction of the ocular surface, PHEMA

Zainuddin et al. / Radiation Physics and Chemistry 80 (2011) 219–229220

hydrogels have been modified by a variety of methods. In earlierwork (Zainuddin et al., 2008), the PHEMA surfaces were modifiedby graft polymerization of a phosphate-containing monomer viaatom transfer radical polymerization (ATRP). This led to asignificant enhancement of the attachment and growth of humancorneal limbal epithelial (HLE) cells on the modified surfaces,probably due to the changes in surface chemistry and, to someextent, the resulting micro-topography of the surface. In thepresent study, to alter the surface chemistry and the surfacetopography of PHEMA hydrogels we have exposed the hydrogelsurfaces to 157 nm F2 excimer laser radiation.

Excimer or exciplex lasers are pulsed lasers that operate withrare gas molecules (Ar, Xe, Kr), or reactive gas molecules (F2, Cl2),or their combination as the gain medium to provide intense,coherent UV and VUV radiation. The ability of excimer laserradiation to remove material in a highly controlled fashion fromthe surface of solid organic matter was discovered in the early1980s (Kawamura et al., 1982; Srinivasan and Leigh, 1982). Theprocess of spontaneous cleavage and ejection of matter caused bythe radiation is termed ablative photodecomposition, and is alsoreferred to as photoablation or, more generally, as ablation. For apolymer to undergo ablation it must contain a chromophore thatwill absorb the incident radiation in order to initiate the ablationprocess. This chromophore might be a characteristic of thepolymer or an adventitious impurity present in the polymer. Itmight also be a product of a photochemical reaction produced atthe polymer surface as a result of an initial absorption of theincident radiation by another chromophore.

Any polymeric material exposed to photons of an appropriateenergy (wavelength) greater than E3.6 eV (lo345 nm) that canbe absorbed may undergo photochemical excitation leading todegradation, with the subsequent formation of new chemicalspecies. If these species are of low molar mass and formed at thepolymer surface, volatile fragments may escape (ablate) from thesurface. The degraded polymer surface may then undergo furtherphotochemical or secondary chemical reactions, such as oxida-tion, crosslinking or other reactions dependent on the surround-ing medium. The surface ablation process has been shown tooccur much more rapidly when a polymer is exposed to laserradiation of an appropriate wavelength with an energy density orfluence (J cm�2) above a particular threshold value. This thresh-old fluence has been shown to be dependent on the chemicalstructure of the polymer and the wavelength of the laser beam.

Often these photochemical reactions are accompanied byphotothermal processes in which the absorbed energy is dis-sipated to surrounding molecules, so raising the local temperatureand possibly causing surface melting and/or charring of thepolymer. This is much more likely to happen when the fluence ofthe radiation is high, as is the case for laser radiation. A moredetailed description of all of the above processes can be found inthe literature (Srinivasan and Braren, 1989; Chirila and vanSaarloos, 1992; Costela et al., 1995a; Sato and Nishio, 2001).

The ablation of hydrated polymer gels (hydrogels) has beenalso investigated (Tsunoda et al., 1999, 2000; Feltham andStapleton, 2002; Dair and Chirila, 2003). Interestingly, all of theseauthors found that the ablation rate was consistently higher in thehydrated state compared with the dry state, even though theablation rate was lower for increasing water content of the gel.These findings certainly imply that the water in the hydrogels hasan influence on the ablation process.

There have been no previously reported studies of the ablationand surface modification of hydrogels using VUV radiation in theregion where water absorbs. Herein we discuss our observationsregarding the changes in the surface chemistry and surfacetopography of PHEMA hydrogels induced by 157 nm laserradiation, in which we believe that the water and its radical

photolysis products play a major role in the ablation process. Thegrowth of human corneal epithelial cells on these modifiedsurfaces has also been studied.

2. Materials and methods

2.1. Materials

2-Hydroxyethyl methacrylate (HEMA) and ethylene glycoldimethacrylate (EGDMA) were purchased from BIMAX (USA)and were used without further purification. Ammonium persul-fate (APS) and ascorbic acid (AA) were supplied by Sigma-Aldrichand used as received. Deionized water was used as a solvent.Unless otherwise specified, all materials and agents for biologicalstudies were supplied by Sigma-Aldrich (USA).

2.2. Preparation of hydrogels

PHEMA was synthesized according to the procedure describedby Zainuddin et al. (2008). HEMA monomer, in the presence of30 wt% water, 0.35 wt% EGDMA, as crosslinking agent,0.185 mmole APS as initiator and an equivalent number of molesof AA (to APS), was polymerized between silanized glass platesspaced separated by 1–2 mm at 50 1C for 24 h. After polymeriza-tion, the polymer hydrogel sheets were extracted for 2 days in aSoxhlet extractor and discs of 7 mm diameter were cut and storedin deionized water. At equilibrium the water content of thePHEMA hydrogel is 38.4 wt%.

2.3. Laser treatment

Before placing the PHEMA hydrogel disc between two CaF2

discs for photolysis, the water on the surface of the sample wasremoved by gently wiping with lint-free tissue paper. The discswere individually exposed to a uniform pulsed laser beam (beamarea 0.5 cm2) from a fluorine (F2) excimer laser (Lambda Physik,Optexs) operating at 157 nm at room temperature under anitrogen environment ([O2]o50 ppm). The laser energy was setto a value of 0.4 mJ/pulse and the numbers of pulses accumulatedwere 1000 and 5000 to yield exposure energy doses of 0.4 and 2 J,(equivalent to fluences of 0.8 and 4 J cm�2), respectively.

2.4. Light microscopy (LM)

Prior to LM observation the samples were rinsed withdeionized water and soaked in water. Optical images wereacquired with a THALES OPTEM 100C Series 10:1 Zoom OpticalSystem fitted with a Carl Zeiss 40� /0.60 objective and 10 mmfibre optic adapter, all assembled on a frame which had beenpurpose-built for simultaneous AFM imaging and optical mea-surements. The source of incident light was an EKE 150 W lamp(ellipsoidal dichroic reflector) with transmission from 400 to700 nm. Optical images were collected using EPIX XCAP V2.1software.

2.5. X-ray photoelectron spectroscopy (XPS)

The cryo-XPS spectra of frozen hydrogel samples wereacquired using a Kratos Axis ULTRA XPS spectrometer incorporat-ing a 165-mm hemispherical electron energy analyser. The sourceof X-ray incident radiation was a monochromatic Al Ka(1486.6 eV) at 150 W (15 kV, 10 mA). Survey (wide) scans weretaken at an analyser pass energy of 160 eV.

Zainuddin et al. / Radiation Physics and Chemistry 80 (2011) 219–229 221

2.6. Attenuated total reflectance—Fourier transform infrared

spectroscopy (ATR-FTIR)

The infrared spectra of PHEMA hydrogel samples wereacquired in the region 525–4000 cm�1 using a Nicolet 5700 FT-IR with a diamond crystal for the ATR objective, at a measurementresolution of 4 cm�1 and with accumulation of 64 scans.

2.7. Atomic force microscopy (AFM)

The surface topography images and roughness measurementsof the hydrogel samples were performed using a Nanoscope IVmultimode scanning probe microscope (VEECO Instrument Inc.).All AFM measurements were conducted in tapping mode using acommercial pyramidal tip (RTESP7, VEECO Instruments) on anetched Si-cantilever with a resonant frequency of 320 kHz and anominal force constant of 42 N m�1.

2.8. Cell culture and attachment assay

Human limbal tissue was obtained with prior donor consentand institutional ethics approval, and processed by establishedtechniques in order to isolate limbal epithelial cells (Barnard et al.,2001). Briefly, donor corneoscleral rims were washed for 5 min inPBS 3 times before being placed into 25 caseinolytic units/mL ofdispase for 1 h at 37 1C. The limbal epithelial cells were thendislodged and collected into excess PBS using a 100 mL pipette.The resultant cell suspension was centrifuged at 300g for 5 minthen the supernatant was discarded with the pellet beingresuspended in 1 mL of TrypLE (Invitrogen Australia Pty. Ltd.)and incubated for 5 min at 37 1C. Excess PBS was then added todilute the TrypLE and the suspension was centrifuged at 300g for5 min. The supernatant was again discarded and the cell pelletwas resuspended in PCT Corneal Epithelium Medium (Chemicon,Boronia, Australia) and then seeded into a T25 culture flask. Onceconfluent, the HLE cells were passaged by briefly washing the cellswith Versene (Invitrogen Australia) followed by incubation inTrypLE at 37 1C for 10 min. The cells were seeded both ontohydrogels and tissue culture plastic (TCP) dishes as a standardwith a cell density of 3.125�104 cm�2. The cells were grown for5 days before being fixed with 3.7% paraformaldehyde. Thesubstrates were stained with 160 nM phalloidin for 25 min toreveal the actin cytoskeleton of the attached cells and then with300 nM DAPI for 5 min to highlight cell nuclei. Four random fieldsof each substrate were photographed and the attached fluores-cently stained cells were counted. Differences in cell counts wereconsidered significant at p-valueo0.05.

3. Results and discussion

3.1. Ablation of dry PHEMA

UV, deep UV and VUV laser ablation of PHEMA in the dry statehas been reported previously by Chirila and Van Saarloos (1992),and Vernon et al. (1992), at 193 nm; Dair and Chirila (2003), at213 nm; Costela et al. (1995a), at 308, 222 and 193 nm andCostela et al. (1995b), at 157 nm. In the UV region and the deepUV at 193 nm the absorption of the incident radiation results fromthe presence of relatively weakly absorbing chromophores inPHEMA. For example, Shirai et al. (1999), have reported thepresence in poly(alkyl methacrylate)s of a broad absorption at215 nm with a peak absorbance of 0.19 mm�1. The absorption oflaser radiation and the subsequent ablation of dry PHEMA at 193,213 and 222 nm could be dependent on the presence of a similar

absorption band in PHEMA. Shirai et al. (1999), have also shownthat as the photolysis of a range of methacrylates polymers undervacuum at 185 nm proceeds, a new intense and very broadabsorption band builds up at 195 nm. They demonstrated thatthis absorption eventually dominates the absorption spectrumover the 190–400 nm range as the ablation process proceeds.The authors believe that this new absorption band is dueprincipally to the formation of olefinic bonds in the polymerduring photolysis, resulting from main-chain scission. Thesesecondary species could play an important role in the ablationof polymethacrylates at 193 nm and longer wavelengths.

As distinct from the behaviour of PHEMA at 193 nm and longerwavelengths, at wavelengths below E190 nm dry PHEMA beginsto absorb VUV radiation very strongly (Cefalas et al., 2000), and at157 nm it has an absorbance of 6.5 mm�1. This very highabsorbance means that 157 nm photons incident on dry PHEMAwill be essentially all absorbed within less than 1 mm of thePHEMA surface. As a result of the high absorbance of PHEMA,Costela et al. (1995b) have concluded that ablation at 157 nm isdifferent from that at longer wavelengths, and could bepredominantly a direct photochemical decomposition process(Lippert, 2005). Costela et al. (1995b) also found that the fluencerequired to produce a given etch depth at 157 nm wassignificantly lower than that needed in the UV region.

3.2. Ablation of PHEMA hydrogels

Feltham and Stapleton (2002) have reported a study of the roleof water in ablation of PHEMA hydrogels exposed in the deep UVat 193 nm and Dair and Chirila (2003) have studied the laserablation of PHEMA hydrogels at 213 nm. Feltham and Stapletonfound that there was a change in the water content of the PHEMAgels during ablation at 193 nm and that the ablation rate wasgreater than that for the dry polymer. Dair and Chirila also foundthat in the presence of water the ablation rate increased over thatfor the dry state, even though water does not absorb radiation ofthis wavelength. Dair and Chirila reported that the ablation ofPHEMA hydrogels at 213 nm was more complex than theyexpected. They proposed that heat transfer to water as a resultof the PHEMA photolysis, producing explosive vaporization ofwater or the formation of rapidly expanding and contractingwater vapour bubbles, might be responsible for the enhancementof the ablation rate for the hydrogels. A similar mechanism toexplain the loss of material from hydrogels has been proposed byTsunoda et al. (2000).

These earlier studies of the laser ablation of PHEMA in the dryand wet states at wavelengths where water does not absorb raisethe question as to what role water might play in the ablation ofPHEMA in the VUV region, where water does absorb very strongly.

3.3. Surface chemistry after ablation at 157 nm

Typical results from cryo-XPS surface analyses of a 157 nmlaser-exposed surface (fluenceE4 J cm�2) and of an unexposedsurface of a PHEMA hydrogel are presented in Fig. 1. It can be seenfrom the XPS survey spectra shown in Fig. 1A that the laserirradiation has led to an increase in the intensity of the C 1s peakrelative to the intensity of the O 1s peak. The C/O uncorrectedpeak intensity ratios obtained from the spectra are: unexposedcontrol 0.74 and exposed surface 0.97. It should be noted herethat the XPS analysis probes the hydrogel surface to a depth ofapproximately 5–10 nm, so the elemental composition of thesurface is an average over this depth. The change in the C/O ratioimplies that under the action of the laser radiation, O-containing

Zainuddin et al. / Radiation Physics and Chemistry 80 (2011) 219–229222

fragments are preferentially lost from the side chains of thePHEMA molecules.

In order to verify this conclusion, and gain further informationabout the chemical changes that take place, the high resolutionC 1s and O 1s XPS spectra have been acquired, as demonstrated inFig. 1B and C. These high resolution spectra were fitted tocomponent peaks according to reported element binding energies(BE), where the bonding of the primary and secondary atoms hasbeen taken into account (Beamson and Briggs, 1992). The peak fitsand percentage contributions for the various structural compo-nents are presented in Fig. 2 and Table 1, respectively. In Fig. 2,the C 1s peaks 1 and 2 are due to the main-chain and methylcarbons (BE¼284–286 eV). The C 1s peaks labelled 3, 4 and 5(BE¼286–290 eV) are due to the three ester side-chain carbonatoms bonded to oxygen, with peak 5 being the ester carbonyl

Fig. 1. XPS spectra of unexposed (control), solid line, and laser-exposed PHEMA

hydrogel surface (fluence 4 J cm�2), dotted line: (A) survey scan, (B) high

resolution C 1s and (C) high resolution O 1s.

Fig. 2. Peak fit (solid line) of high resolution XPS spectra of C 1s and O 1s for unexposed

hydrogels. The experimental spectrum is the dotted line.

carbon and peak 3 being the other side-chain ester group carbonthat is bonded to oxygen. These peak analyses for the unexposedcontrol and the exposed surfaces confirm that the contributions ofthe carbon atoms associated with the C 1s peaks 1 and 2 increasedin intensity (number) after exposure to the laser, relative to theintensities of the C 1s peaks due to the ester side-chain carbons.Thus there is a loss of ester groups from the polymer chains in thenear-surface layers of the PHEMA hydrogel. However, it is notpossible to assign the observed increase in the peak 1 and 2contributions in the BE¼284–286 eV region to any specific typesof aliphatic carbons, as XPS measurements cannot resolve thedifferences between C–H, C–C and CQC bonds.

The peak fitting of the oxygen O 1s peak (BE¼531–535 eV)(Fig. 2) revealed the presence of three component peaks, due tothe three ester side-chain oxygen atoms. Before exposure thecontributions of the three components were found to be equal,but after exposure to the laser radiation, the contribution ofcomponent 1, due to the ester carbonyl oxygen, appeared toincrease slightly relative to the other two side-chain components

(control) and laser-exposed fluence 4 J cm�2 (dose 2 J, exposed are 0.5 cm2) PHEMA

Table 1Peak fitting results of high resolution XPS (C 1s and O 1s) for unexposed (control)

and laser-exposed fluence 4 J cm�2 (2 J dose, exposed area 0.5 cm2) PHEMA

hydrogels.

Unexposed (control) Exposed 2 J

Number BE (eV) % Number BE (eV) %

Carbon (C) component

1 285.02 41.62 1 285.01 50.65

2 285.75 15.23 2 285.75 15.01

3 286.70 14.81 3 286.65 10.10

4 287.08 14.24 4 287.05 09.58

5 289.17 14.10 40 287.92 02.62

5 289.17 12.04

Oxygen (O) component

1* 532.12 33.51 1* 532.12 34.96

2* 532.81 33.17 2* 532.80 31.24

3* 533.65 33.32 3* 533.67 33.80

Zainuddin et al. / Radiation Physics and Chemistry 80 (2011) 219–229 223

(see Table 1). This suggests the presence of some new doublybonded oxygenated species in the exposed surface, perhapsresulting from a loss of the ethylene glycol components of theester groups.

The peak fits for the C 1s peak after exposure to the laserrequired the inclusion of peak 40 at BE¼287.9 eV in order toobtain a satisfactory representation of the overall peak. A similarpeak was observed by Hozumi et al. (2002), in the XPS spectra of adry poly(methyl methacrylate) sample after exposure to a 172 nmlaser in the presence of a low pressure of oxygen. They haveassigned this peak to the formation of carbon atoms with twobonds to oxygen. This would be consistent with the increase inthe contribution of peak 1 in the curve fit of the O 1s peak for theexposed PHEMA hydrogel surface. Such a CQO peak could ariseas a result of the formation of aldehyde groups, for example.

Further information about the chemical changes in theexposed PHEMA surface was obtained from FT-IR/ATR analyses.An ATR analysis using a diamond internal reflection elementprobes the surface layer of the hydrogel to a depth of up to abouttwo micrometers, so penetrating much more deeply than the XPSanalysis. Typical ATR spectra over the range 4000–500 cm�1 areshown in Fig. 3 for the control and for incident exposure fluencesof approximately 0.8 and 4 J cm�2. Apart from a decrease in therelative intensity of the OH bands at 3300 and 1650 cm�1, therewas little other visible significant change in the spectra over thewavelength range delineated. This observation is consistent withthere being little change in the structure of the PHEMA averagedover the depth range of the ATR analysis. While the XPS spectradid show a change in the oxygen to carbon ratio over a depth of5–10 nm, it was clear from the XPS spectra that many of theHEMA residues in this region were still intact, even at a fluence of4 J cm�2.

These observations clearly suggest that the most extensivelydamaged surface material was probably lost (ablated away)during the laser exposure treatment. They also indicate that mostof the chemically modified material remaining after exposure isprobably limited to a depth of about 50 nm from the hydrogelsurface, where most of the incident photons will have beenabsorbed. This is expected from the large absorbance cross-sections of water and PHEMA at 157 nm, and is discussed furtherbelow.

The change in the intensity of the OH peaks in the ATRspectrum is indicative of a change in the hydrophilicity of thesurface layers of the hydrogel, and is consistent with an increasein the carbon-to-oxygen ratio near to the exposed hydrogelsurface, rendering the surface more hydrophobic. It is alsoconsistent with observations made after laser exposure of PHEMAhydrogels at longer wavelengths (Feltham and Stapleton, 2002),that showed for ablation at 193 nm, that the water content waslower in the exposed hydrogel.

Fig. 3. ATR-FTIR spectra of an unexposed (control) and laser-exposed PHEMA

hydrogels for incident fluences of 0.8 and 4 J cm�2 (doses of 0.4 and 2 J and

exposed area 0.5 cm2).

3.4. The role of water in the 157 nm laser ablation process

Although water does not absorb in the UV or deep UV region, itabsorbs very strongly in the VUV region (Mota et al., 2005;Azrague et al., 2005), with the so-called first absorption band forwater extending from approximately 6.6 eV (190 nm) to 8.6 eV(145 nm). At 157 nm the absorption cross section for water is3.0 Mb, equivalent to an absorbance of E10 mm�1, and theabsorption is dissociative (Mota et al., 2005). So photolysis ofwater at 157 nm leads to the direct formation of hydrogen andhydroxyl radicals.

H2O-Hd+OHd

The absorbance of water is much greater than that for PHEMAat 157 nm (10 mm�1 compared to 6.5 mm�1). In addition, theequilibrium water content of the PHEMA hydrogel is E38.4 wt%(Zainuddin et al., 2008), which corresponds to a mole ratio ofwater to HEMA of 4.4–1.0. Therefore, when a saturated PHEMAhydrogel is exposed to the 157 nm laser radiation, the waterpresent within E1 mm of the exposed hydrogel surface willabsorb almost all of the incident photons. This means that thePHEMA present in this region of the gel will be exposed to a highflux of hydroxyl and hydrogen radicals. The hydroxyl radicals inparticular will efficiently abstract hydrogen from the PHEMA,leading to the formation of polymer chain radicals. The polymerradicals formed during photolysis will subsequently undergo avariety of scission and other reactions leading to the loss andfragmentation of ester side-chains, scission of the PHEMAmain chains and the formation of new polymer chain structures(e.g. Hill et al., 1996). There would be an overall loss of oxygenatoms relative to carbon atoms and this would create a morehydrophobic region near the exposed surface.

This implies that the processes responsible for the ablation ofPHEMA hydrogels at 157 nm, at least initially, differ significantlyfrom those that have been proposed to explain ablation at longerwavelengths. While much of the energy deposited in the PHEMAhydrogel surface will be lost to the small PHEMA and waterfragments formed during photolysis, some energy will also bedissipated to the hydrogel locally, and that could result in thevaporization of water molecules. However, it is worth noting thatthere was no evidence for the presence of transient melting of thePHEMA at the surface or for crater formation (see later), as hasbeen reported in some cases of laser ablation of PHEMA at longerwavelengths (for example, Costela et al., 1995a). It is also worthnoting that the hydrogel samples remained clear and hydratedafter photolysis.

Although the water molecules are much more likely to absorbthe photons incident on the hydrogel surface, some photons will beabsorbed by the PHEMA. In addition, if the outermost exposedsurface layers of the hydrogel become depleted in water, eitherthrough radical formation or evaporation, then it is also possiblethat direct photolysis of the PHEMA could become important inwater-depleted regions. However, because the absorbance ofPHEMA is also very high, though not nearly as high as for water,the incident photons will still only penetrate a very short distanceinto the hydrogel. Thus, the photochemistry which occurs as aresult of exposure of a PHEMA hydrogel to 157 nm laser radiationwill be limited to just the near-surface region of the hydrogel,i.e. probably principally to a depth of less than 50 nm below thesurface, since the photon flux will decay exponentially with depth.

Mechanisms for direct photochemical decomposition ofmethacrylates are well established, and they are known todegrade mainly via the so-called Norrish mechanisms (Hartleyand Guillet, 1968). The Norrish processes have been summarizedin Fig. 4, and they too predict the loss and fragmentation of themethacrylate ester side-chains (Norrish type I mechanisms) and

Fig. 4. Proposed Norrish-type mechanism for side and main-chain laser-induced cleavage of PHEMA following direct absorption of radiation at 157 nm.

Zainuddin et al. / Radiation Physics and Chemistry 80 (2011) 219–229224

polymer chain scission (Norrish type II mechanism). So theseprocesses would also contribute to making the exposed surfacemore hydrophobic.

It is not possible to rule out a role for explosive vaporizationof water or the creation of collapsing water vapour bubbles(for example, Dair and Chirila, 2003) in the ablation of PHEMA at157 nm. However, these processes could be less important at157 nm because of the very high absorbance of the water in thenear-surface hydrogel layers, meaning that any vaporized materi-al may readily escape from the matrix surface. Likewise, theabsorbances of the secondary products of photolysis (Shirai et al.,1999) may also be less likely to play a critical role in the ablationbecause of the very high molar absorptivities of the water andPHEMA at 157 nm.

One further important role of water that should not beoverlooked is its plasticization of the polymer in a hydrogel.PHEMA is a glassy solid at room temperature, while fully hydratedPHEMA is soft and pliable. As a result diffusion of species andpolymer chain motions are significantly enhanced in the hydrogelrelative to PHEMA in the dry state. Consequently, the yields ofsome PHEMA degradation reactions will be greater in thehydrogel compared to the dry state. For example, as a result ofthe increased PHEMA chain and fragment conformational free-dom and mobility in the hydrogel, the probability for geminaterecombination of scission radicals will decrease, thus leading tohigher yields for permanent scission. Dong et al. (1995) haveshown that for the gamma radiolysis of PMMA at 353 K in the

solid state the yield of permanent chain scission is E2.0 but theyield of temporary chain scission is much higher at 18.6, thedifference being due to geminate radical recombination reac-tions.) So the plasticizing role of water in hydrogels could accountfor some of the observed differences in polymer etch ratesbetween dry polymers and hydrogels.

3.5. Surface topography

Fig. 5 shows the surface topography of the unexposed (control)and laser-exposed PHEMA hydrogels as measured by lightmicroscopy and AFM. As expected, the unexposed samples(Fig. 5A0–C0) show low roughness with the largest topographicalfeatures being �50 nm in height (Fig. 5C0). Typical fields of viewshow raised regions in the form of straight lines (50–85 nm high)due to defects on the polymer surface generated by the scratcheson the glass plates that were used as moulds when preparing thepolymer sheets. The surface topography of the exposed samples(Fig. 5A0.4 J–C0.4 J and A2 J–C2 J) shows rippled structures withnano-scale roughness perpendicular to the surface that increaseswith increasing exposure dose. Typically, for the samples exposedto an incident fluence of 0.8 J cm�2 (dose¼0.4 J) the surfacefeatures were of 50–120 nm in height and 2–5 mm in width(distance peak-to-peak) (Fig. 5C0.4 J), so the aspect ratio for thesefeatures is very low. For an incident fluence of 4 J cm�2 (dose¼2 J)the surface roughness increased with the features being

Fig. 5. Surface topography of unexposed and laser-exposed PHEMA hydrogels: (A) LM images (scale bars: 50 mm), (B) AFM images (scale bars: 20 mm) and (C) surface

roughness.

Zainuddin et al. / Radiation Physics and Chemistry 80 (2011) 219–229 225

approximately 250–500 nm in height with a width of 3–10 mm(Fig. 5C2 J). Again, the aspect ratio for the features is very low. Theexposed surface profiles show no evidence for deep craterformation in the etched area or of a build-up of debris aroundthe exposed region. These observations are consistent with therebeing no explosive removal of large fragments of surface material.

3.6. Review of previous studies of cell attachment and growth on

ablated polymers

It is important to know which of the changes induced by laserablation of polymers has the predominant effect on theirbioadhesivity: chemical or physical (i.e., the topography). To date,only limited experimental work has been carried out to elucidatethis issue. Fewster et al. (1994) were probably the first to attemptlaser ablation of synthetic polymers with the aim of improvingcell adhesion and biocompatibility, but they limited their studyonly to the topographic aspects. Polystyrene (PS), poly(ethyleneterephthalate) (PET) and poly(methyl methacrylate) (PMMA)were ablated with the 248-nm radiation emitted by a KrF excimerlaser. The force of adhesion of epithelial cells and osteoblasts wasmeasured and about 5% enhancement was observed on theablated surfaces. It was concluded that cells prefer to adhere atthe edges and pillars generated by laser exposure.

In a similar study (Hunt et al., 1995), polycarbonate (PC) andpolyetherimide were ablated with 248 nm laser radiation. Thebehaviour of human neutrophils was monitored by tracking theirshape change and speed of movement as compared to cellsstimulated by weak or potent chemotactic agents. The cellsremained in an unstimulated state on the ablated surfaces to alevel even lower than in the presence of weak stimulators.

In another study (Dadsetan et al., 2001), PET was exposedto radiation emitted by two types of lasers, an UV excimer laser

(KrF, emitting at 248 nm wavelength) and a CO2 laser (emitting inthe near infrared at 9250 nm), and the behaviour of murine L929fibroblast cells on the exposed and unexposed polymer surfacewas examined after 48-h incubation. On surfaces ablated by theexcimer laser, spreading of cells was statistically more significantthan that on untreated surfaces. However, by increasing thenumber of laser pulses, spreading of cells decreased while thewettability of the surface (estimated by contact angle measure-ments) started to increase. Formation of hydrophobic regions onthe surface was suggested as a result of laser-induced chemicalchanges. The topographic micropatterning of the surface appearedto be less important in the case of the excimer laser ablation(1 mm or less) as compared to the exposure to CO2 laser (at least10 mm). Generally, the treatment of PET surface with the infraredlaser radiation led to a direct interdependence between number ofradiation pulses, wettability and cell spreading, while thiscorrelation was rather ambiguous in the samples ablated byexcimer laser.

An excimer laser emitting at 193 nm (ArF) was used byBremus-Kobberling and Gillner (2003) and Bremus-Kobberlinget al. (2004) to ablate a large number of commercial polymers.The polymers included PMMA, PC, PS, PET, poly(etheretherketone)(PEEK), perfluoroalkoxy copolymer (PFA), a cycloolefin copolymerand poly(dimethylsiloxane) (PDMS). Both changes in the wett-ability of the surface (in terms of hydrophilicity/hydrophobicity,estimated by contact angle measurements) and the inherentformation of micropatterns on the surface were suggested asfactors able to influence the biological properties of the surfaces.Different topographies were created, including ripples, scales,wells and pillars, within a large range of dimensions(0.25–50 mm). The researchers discovered that by increasing theroughness of the ablated surface due to creation of topographicfeatures, the pre-existing wetting characteristics are enhanced inthe same direction, i.e. a hydrophilic surface becomes

Zainuddin et al. / Radiation Physics and Chemistry 80 (2011) 219–229226

more hydrophilic, while a hydrophobic surface becomes morehydrophobic (‘‘superhydrophobic’’).

The interdependence between surface roughness, water repel-lence and reduction of the contaminating particle adhesion has beenobserved in the plant kingdom and coined the ‘‘lotus-effect’’(Barthlott and Neinhuis, 1997). The rougher a surface, the morehydrophobic it becomes. Bremus-Kobberling’s group demonstratedsuch an effect in the case of ablated PEEK (hydrophilic) and PDMS(hydrophobic). However, they investigated cell adhesion to only oneof the polymers (PDMS), using murine L929 fibroblast cells. Itappeared that on the ablated surface cells tend to grow preferen-tially into the spatial features created by ablation (ripples, etc.).

A study of the effects of 193-nm excimer radiation (Sherlocket al., 2003) focused on the effect of induced chemical changes in theablated layer, rather than on topographic changes on the surface. Acommercial polyamide (Nylon-12) and PET (as Dacron) were bothablated with an ArF excimer laser (193 nm), and then the surfaceswere investigated by XPS, ATR-FTIR and Raman spectroscopies, andby contact angle measurements. The deposition of fibronectin, anadhesion protein, was also carried out by incubation and estimatedby antibody/specific fluorophore treatment followed by laserscanning confocal microscopy. The adhesion and growth of cultured3T3 fibroblast cells was evaluated on both the ablated andunexposed polymer surfaces. XPS indicated an increase in theoxygen content of the modified layer, but the other spectroscopictechniques could not detect any changes. The hydrophilicity of thesurface, estimated by the value of contact angle, increased followingablation in a very definite manner. On the ablated surfaces, the cellsattached, spread and developed a cytoskeleton, while on theunexposed samples they adopted a rounded morphology. This resultcontradicts the general opinion that hydrophilic polymers do notsustain cell adhesion (Lydon et al., 1985; Lydon, 1986). An evenmore surprising result of this study was that the same amount offibronectin was adsorbed on both the ablated and unexposedsurfaces, yet the cell attachment and spreading on the ablatedsurfaces was considerably enhanced. This would suggest that thedeposition of proteins is not a prerequisite for cell adhesion, thusapparently discrediting a fundamental tenet in this field (Horbettand Klumb, 1996). Aware of this discrepancy, these investigatorssuggest that a correlation between adsorption of fibronectin andsurface energy (as expressed by the contact angle) may not be valid,confirming an earlier suggestion (Vogler, 1998).

Non-laser irradiation with high-energy UV emitted by a lowpressure lamp (185 and 253 nm) was also used to expose polymerssuch as PMMA, PS and PC (Welle and Gottwald, 2002). Formation ofperoxide and carboxyl groups was detected on the surface, togetherwith an increase of hydrophilicity. The attachment of two differentcell lines (HepG2 and L929) in culture was assessed on PS and itincreased significantly after UV exposure. However, in the case ofPMMA and PC, the cells attached in greater numbers on theunexposed surfaces, and a possible pre-existing contamination ofthe commercial samples was considered responsible for this finding.As the investigators did not attempt a thorough cleaning of thesesamples, the results can only be regarded as inconclusive.

Thus to further extend these previous studies on dry polymerswe have examined the cell attachment and growth on the 157 nmlaser-ablated surfaces of the PHEMA hydrogels.

3.7. Surface bioactivity and HLE cell attachment and growth on the

PHEMA hydrogels

3.7.1. Cell response to surface chemistry

We have demonstrated that the surfaces of the PHEMA hydrogelsfollowing exposure at 157 nm were modified chemically, containrelatively fewer ester side chains and display greater surface

roughness. Also, the near-surface layers are somewhat less hydratedthan the surfaces of the unexposed hydrogels or of the bulk matrixof the exposed hydrogels. These changes appear to bring about animprovement in cell attachment, spreading and growth on the laser-exposed surfaces. As shown in Figs. 6 and 7, the laser-exposedsamples display better cell attachment and growth as compared tothe unexposed samples (p-valueo0.05). The number of cellsattached and proliferated on the samples exposed to an incidentfluence E4 J cm�2 (dose¼2 J) was higher than those found on theunexposed control and on commercial TCP dishes.

Cell attachment occurs as a result of an intimate interactionbetween the surface of a substrate and the extracellular matrix(ECM). The ECM contains a diverse array of functional groups (e.g.hydroxyl, glycoside groups in polysaccharides and amide groups inglycoproteins), as well as highly ordered rigid segments (e.g. elastinand collagen segments) of the ECM proteins. None-the-less there isstill great uncertainty in elucidating the details of cell attachment onsurfaces having well-defined chemistry, such as in the case ofsynthetic polymers. Nevertheless, it can be stated that a significantimprovement in cell attachment and growth was observed for laser-exposed PHEMA hydrogels that may be associated with a reductionof the surface hydrophilicity and the appearance of some moreheterogeneous surface chemistry, along with the development ofsurface topography. A number of reports have demonstrated thathydroxyl groups are stimulatory to cell adhesion and growth whenthey are in low/moderate density and/or randomly distributed, orwhen they are assembled on hydrophobic materials. For example, itwas reported (Horbett et al., 1985; van Wachem et al., 1987) that thecell-adhesive properties of HEMA-based copolymers steadily in-creased with an increasing fraction of ethyl methacrylate (EMA) ormethyl methacrylate (MMA) in the copolymers. Margel et al. (1995)studied the influence of various terminal functional groups, such as–CF3, –CH3, –CO2CH3 and –CH2OH, on self-assembled monolayers onsilicone and glass surfaces, and found that endothelial cell attach-ment, as well as improved growth in the order –CH2OH4–CO2CH34–CH34–CF3. It was also shown (Lee et al., 2006) thaterythroleukemia cells attached more readily on hydroxyl-functio-nalized silicon wafer surfaces as compared to the surfaces modifiedwith NH2 and CH3 groups. In a study (Owens and Gingell, 1988) ofthe role of OH groups on the attachment of amoeba cells, it wasclaimed that a long-chain alcohol (C22H45OH) in a two-dimensionalarray with a known density, provided a surface that was stronglyadhesive for cells. Different forms of oxygen have also been reportedto influence cell attachment and growth (Rubin, 1966; Ramsey et al.,1984; Tegoulia et al., 2001; Sherlock et al., 2003). In pioneering work(Rubin, 1966), the surfaces of Petri plates made of polystyrene (PS)were rendered cell-adhesive through surface oxidation withconcentrated sulphuric acid. The cell adhesivity was attributed tonegatively charged oxygen in the form of sulphonate (SO3

�) groupsthat induce surface wettability, a term which is commonly used todescribe the balance of hydrophobicity and hydrophilicity of thematerials (Garcia, 2008). Nowadays, the majority of commercialtissue culture plastic, including PS, poly(ethylene terephthalate)(PET) and poly(methyl methacrylate) (PMMA), are treated with anoxygen plasma or corona discharge to generate surface wettabilityand it is surmised that the enhanced bioadhesivity is a result of thepresence of oxygen, mainly as carboxylate (COO�) groups. Otheroxygenated structures which have also been identified to promotecell attachment are CH2OH, COCH3, CO2CH3, CONH2 and CO2NH2

(Ertel et al., 1991; Margel et al., 1995; McClary et al., 2000; Tegouliaet al., 2001; Spargo et al., 2005; Roach et al., 2007).

3.7.2. Cell response to nanotopography

The number of cells that attached and grew on the samplesexposed to a fluence of 4 J cm2 was significantly higher

Fig. 6. Fluorescence micrographs of HLE cells after 5 days of culture on unexposed (A) and laser-exposed PHEMA hydrogels for incident fluences of 0.8 J cm�2 (dose of 0.4 J,

exposed area 0.5 cm2) (B) and 4 J cm�2 (dose 2 J, exposed area 0.5 cm2) (C), and for unexposed PS tissue culture plastic (D). Scale bars: 100 mm.

Fig. 7. Attachment and growth of HLE cells after 5 days of culture on unexposed

and laser-exposed PHEMA hydrogels (doses¼0.4 and 2 J, exposed area 0.5 cm2),

and on PS tissue culture plastic (TCP). A single asterisk indicates a p-valueo0.05,

while double asterisk indicates a p-valueo0.001.

Zainuddin et al. / Radiation Physics and Chemistry 80 (2011) 219–229 227

(p-valueo0.05) when compared to the number of cells thatattached and grew on the samples exposed to a fluence of0.8 J cm�2, or on a smoother (control) unexposed surface(p-valueo0.001) (see Fig. 7). The important role of the surfacetopography of the substrate in cell attachment and growth hasbeen known for a long time (Weiss, 1941; Curtis and Varde, 1964).However, since the initial reports numerous studies have re-emphasized the enhancement in cell attachment observed forsurfaces patterned with topographic features having nanometerto micron dimensions. For example, the works of Singhvi et al.(1994), Curtis and Wilkinson (1998), Flemming et al. (1999),

Teixeira et al. (2003), Karuri et al. (2004), Yim and Leong (2005)and Liliensiek et al. (2006). Our results reported herein areconsistent with these earlier observations.

4. Conclusions

It has been demonstrated that the ablation of PHEMAhydrogels with high-energy radiation using a 157 nm excimerlaser leads to a modification of the hydrogel surface chemistryand surface topography. A key finding was that only the outersurface layers of the hydrogel were affected by the photolysis as aresult of the very high absorbance of the water in the gel. So,unlike the ablation behaviour of the PHEMA hydrogels at UVwavelengths, at 157 nm the water in the hydrogel surface willabsorb the incident photons to produce hydrogen and hydroxylradicals that can abstract hydrogen from the polymer chains, thusleading to their decomposition and ablation. Dry PHEMA alsoabsorbs 157 nm radiation very strongly, but not nearly as stronglyas does the water. However, should the hydrogel surface becomedepleted in water during photolysis, direct photochemicaldecomposition of the PHEMA chains could also occur. Afterablation, the polymer surface layers have been shown by XPSanalysis to have lost some ester side-chains, making the surfaceless hydrophilic, and leading to a lower water concentration at theexposed hydrogel surface.

The 157 nm laser ablation has been shown to be a suitablemethod for enhancing cell adhesion to the surface of PHEMAhydrogels. This effect is likely due to the chemical changes thattake place and to the newly created topological features on theexposed surface. Because of the high absorbance of the PHEMAhydrogel surface at 157 nm, it is possible that a lower fluencewould be required to produce a biologically active surface layeron these hydrogels than would be required at UV wavelengthswhere the absorbance is much smaller.

Zainuddin et al. / Radiation Physics and Chemistry 80 (2011) 219–229228

Acknowledgements

This study was supported in part by an unrestricted grant fromPrevent Blindness Foundation, Brisbane, Australia, through Viertel’sVision. This research was supported in part by the AustralianResearch Council Discovery Projects and Linkage Equipment,Infrastructure and Facilities funding schemes (Project nos.DP0878615 and LE0668517). The assistance provided by Dr. BarryWood (Department of Chemistry, University of Queensland) in theXPS measurements is gratefully acknowledged.

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