I. MICHALJANICOVA et al.: SURFACE PROPERTIES OF A LASER-TREATED BIOPOLYMER
331–335
SURFACE PROPERTIES OF A LASER-TREATED BIOPOLYMER
LASTNOSTI POVR[INE BIOPOLIMERA, OBDELANEGA Z
LASERJEM
Iva Michaljani~ova1, Petr Slepi~ka1, Silvie Rimpelova2, Petr Sajdl3, Václav [vor~ík1
1University of Chemistry and Technology, Department of Solid State Engineering, Technická 5, 166 28 Prague, Czech Republic
2University of Chemistry and Technology, Department of Biochemistry and Microbiology, Technická 5, 166 28 Prague, Czech Republic
3Institute of Chemical Technology, Department of Power Engineering, Technická 5, 166 28 Prague, Czech Republic
iva.michaljanicova@vscht.cz
Prejem rokopisa – received: 2014-10-14; sprejem za objavo – accepted for publication: 2015-05-04
doi:10.17222/mit.2014.260
Structured surfaces allow the application of commonly used polymers to be extended into specialized fields. This paper
describes the construction of surface structures on biopolymer poly(L-lactide) (PLLA), with a method combining krypton
fluoride laser (KrF), excimer laser exposure and thermal annealing. PLLA is a commonly used substrate for medical purposes,
such as implants and tissue matrices, but it still has a number of limitations, which can be eliminated with its modification. This
work is focused on morphological studies and roughness measurements of a structured PLLA substrate using atomic-force
microscopy (AFM) and chemical changes investigated with UV-Vis spectroscopy and X-ray photoelectron spectroscopy (XPS).
Finally, the biocompatibility of the material was tested using a model cell line of mouse embryonic fibroblasts (NIH 3T3).
Using the laser treatment in combination with thermal annealing, we prepared surface layers with various patterns dependent on
the chosen input parameters.
Keywords: biopolymer, excimer laser, nanostructuring, thermal annealing, characterization
Strukturirane povr{ine omogo~ajo raz{iritev uporabe obi~ajnih polimerov tudi na posebna podro~ja. ^lanek opisuje pripravo
povr{inske zgradbe biopolimera poly(L-lactide) (PLLA), s kombinirano metodo, z izpostavitvijo excimer laserju (KrF) in
toplotno obdelavo. PLLA je obi~ajno uporabljena osnova za medicinske namene kot so vsadki in osnove tkiv, vendar so {e
{tevilne omejitve, ki se jih da odpraviti z njihovim modificiranjem. ^lanek je osredoto~en na {tudij morfologije in meritve
hrapavosti strukturirane PLLA podlage, z mikroskopijo na atomsko silo (AFFM) in preiskave kemijskih sprememb z UV-Vis
spektroskopijo in rentgensko fotoelektronsko spektroskopijo (XPS). Preizku{ena je bila tudi biokompatibilnost materiala z
uporabo modelne celi~ne linije embrionskih fibroblastov mi{i (NIH 3T3). Z lasersko obdelavo, v kombinaciji s toplotno
obdelavo, so bile pripravljene plasti na povr{ini z razli~nimi vzorci odvisno od izbranih vhodnih parametrov.
Klju~ne besede: biopolimer, ekscimer laser, nanostrukturiranje, postopek `arjenja, karakterizacija
1 INTRODUCTION
Poly(L-lactic acid) or poly(lactide) is a biodegradable
and bioabsorbable thermoplastic polyester, produced
from renewable sources.1 In medical applications, it is
used in the matrices for tissue engineering, stents,
sutures or in drug delivery systems, but it still has a lot of
limitations, which can be eliminated with its appropriate
modification.
Polymer structuring allows a utilization of ordinary
materials in highly specialized fields. Nanostructured
materials find different applications, e.g., in DNA and
protein sequencing2, in the creation of a suitable synthe-
tic environment for cell growth3 or in the solar-cell tech-
nology.4 Self-organized structures are prepared with a
bottom-up method. A typical example of nanostructuring
is a ripple or dot formation caused by laser irradiation.
The ripples arise due to the interference pattern forma-
tion at a surface and the subsequent response of the
surface.5
Another example of a self-organizing mechanism is
wrinkling instability, which exhibits a variety of surface
patterns. Wrinkles are produced by the residual stress,
which exceeds the critical value.6 Wrinkle patterns are
included, e.g., in tunable optical devices7 or flexible
electronics.6–8
This paper deals with the surface modification of
poly(L-lactic acid) using laser treatment in combination
with thermal annealing. Treated samples were studied
with atomic-force microscopy (AFM), UV-Vis spectro-
scopy, and ARXPS (angle-resolved photoelectron
spectroscopy). Tests of biocompatibility were carried out
with mouse embryonic fibroblasts (NIH 3T3). With this
modification, we prepared various surface patterns with a
wrinkle-like structure.
2 MATERIALS AND METHODS
2.1 Materials and modification
We used biopolymer poly(L-lactic acid) (PLLA, a
density of 1.25 g cm–3, Tg = 60 °C, a crystallinity of
60–70 %, 50-μm-thick foils, supplied by Goodfellow
Ltd., Cambridge, Great Britain).
For the irradiation of PLLA we used a KrF excimer
laser (Coherent Compex Pro 50, a wavelength of 248
nm, a pulse duration of 20–40 ns, a repetition rate of 10
Materiali in tehnologije / Materials and technology 50 (2016) 3, 331–335 331
UDK 621.793:669.058:66.088 ISSN 1580-2949
Original scientific article/Izvirni znanstveni ~lanek MTAEC9, 50(3)331(2016)
Hz). The beam of the KrF laser was polarized linearly
with a cube of UV-grade fused silica 25 mm × 25 mm ×
25 mm with an active polarization layer. For a homo-
geneous illumination of the samples, we used only the
central part of the beam profile by means of an aperture
0.5 cm × 1.0 cm. The samples were mounted onto a
translation stage, being perpendicular to the laser beam.
The chosen pulses were in a range of 100–6000, having
laser fluences in an interval of 6–30 mJ cm–2.
The thermal treatment of the polymers was accom-
plished with a BINDER thermostat. The samples were
heated to 60 °C (the glass-transition temperature of
PLLA) immediately after the laser treatment. After 30
min of thermal annealing, the samples were cooled down
to room temperature (RT).
2.2 Measurement techniques
The surface morphology and roughness of the pri-
stine and modified polymer samples were examined with
the atomic-force-microscopy (AFM) technique using a
VEECO CP II device in the tapping mode. The tapping
mode was chosen to minimize the damage to the sample
surface. A RTESPA-CP Si probe with a spring constant
of 20–80 N m–1 was used. The mean roughness value
(Ra) represents the arithmetic average of the deviations
from the center plane of a sample.
The presence of oxygen and carbon in the PLLA
surface layer was determined with X-ray photoelectron
spectroscopy (XPS). An Omicron Nanotechnology
ESCAProbeP spectrometer was used. The exposed and
analyzed area had a dimension of 2 mm × 3 mm. The
X-ray source was monochromated at 1486.7 eV.
Characteristic O(1s) and C(1s) peaks were searched for.
Atomic concentrations of the elements were determined
with the CASA XPS program using an integrated area of
spectrum lines and relative sensitivity factors, quoted in
the database of CASA XPS.
UV-Vis spectra were measured using a Perkin Elmer
Lambda 25 spectrometer in a spectral range of 190–1100
nm with a bandwidth of 1 nm (fixed).
2.3 Cytocompatibility tests
For cell-culture experiments, we used an adherent
model cell line of mouse embryonic fibroblasts (NIH
3T3) (ATCC, USA). NIH 3T3 cells were cultivated on a
regular basis in high-glucose Dulbecco’s modified Eagle
medium (DMEM, Sigma, USA) supplemented with
stable 2 mM L-glutamine, 10 % fetal bovine serum and
1 % MEM vitamin solution (Invitrogen, USA). The cells
were maintained at standard conditions (37 °C, a 95 %
humidified atmosphere, 5 % CO2). The cells were main-
tained in exponential growth.
The bio-response of individual PLLA samples was
tested. The polymers were first sterilized in 70 % ethanol
for 1 h, air dried, inserted into 12-well plates for cell cul-
tures (VWR, Ø 2.14 cm) and weighted with poly(methyl
methacrylate) cavus cylinders. The samples were seeded
with the NIH 3T3 cells, with a density of 14,000 cells
per cm–2 in 800 μL of a complete DMEM. An identical
batch of cells growing on a polystyrene Petri dish (PS)
was used as a control. The experiments were done in
triplicates.
The cells intended for a fluorescence-microscopy
analysis were fixed and stained. They were washed twice
with phosphate-buffered saline (PBS, pH = 7.4) and
fixed with 1 mL of a 4 % formaldehyde (Thermo Scien-
tific, USA) solution in PBS at 37 °C for 20 min. A
phalloidin-tetramethylrhodamine B isothiocyanate (Sig-
ma, USA) solution in PBS (0.5 μg·mL–1, 10 min) was
used to visualize F-actin; cellular nuclei were stained
with a solution of 4’,6-diaminido-2-phenylindole dihy-
drochloride (DAPI, Sigma, USA) in PBS (0.5 μg·mL–1,
5 min). During and after the staining, the cells were
rinsed twice with PBS to remove the excess of unbound
dyes.
3 RESULTS
Because the laser itself has just a small effect on the
surface morphology and the roughness of PLLA, we
investigated the influence of the laser treatment in a
combination with thermal annealing. We used excimer
radiation followed by thermal annealing, which was
proven to significantly influence the morphology. For the
reverse order of the applied methods, we observed just
insignificant morphological deviations on the samples in
comparison to the samples treated only by a laser beam.
On the contrary, for those samples where the thermal
annealing was the second step of the treatment, the sam-
ple surfaces were rapidly changed. Moreover, for a low
laser fluence and a high number of pulses (6–15 mJ cm–2
and 6000 pulses) the surface roughness was also signifi-
cantly increased.
Figure 1 shows the influence of the applied methods
on the sample morphology. The modification of the sam-
ples at the top of Figure 1 shows a structure practically
identical with the pristine PLLA and the roughness is
also similar to the Ra value of the pristine PLLA, which
was determined as 6.9 nm. By applying different laser
parameters, we prepared different patterns with various
roughness values. An important parameter affecting the
sample roughness is the combination of the laser fluence
and the number of pulses. If the laser energy is too high,
the surface is flattened (30 mJ cm–2, 6000 pulses, Ra =
0.7 nm), but with a lower energy, the surface prepared
can be extremely rough, with a spongious structure
(15 mJ cm–2, 3000 pulses, Ra = 44.7 nm).
The concentration of the surface oxygen was deter-
mined with XPS and the results for the selected samples
are listed in Table 1. According to these results, it is not
possible to conclude that the oxygen concentration
decreases or increases after the modification because the
differences in the concentration were within the stati-
332 Materiali in tehnologije / Materials and technology 50 (2016) 3, 331–335
I. MICHALJANI^OVA et al.: SURFACE PROPERTIES OF A LASER-TREATED BIOPOLYMER
stical error (about 2 %), which could have taken place
during the measurement. It is possible to conclude that
the oxygen concentration decreases towards the surface
of the spongious structure (the plane-surface oxygen
concentration is higher in comparison with the pattern at
the very top). This decrease can be contributed to the
reorientation of dipoles (oxygen-containing groups)
toward the polymer surface.
Table 1: Element concentration of the PLLA surface. The values were
determined, with XPS method, for pristine and laser-treated
(9 mJ cm–2, 6000 pulses) samples, further for samples treated with
laser followed by thermal annealing (9 mJ cm–2, 6000 pulses, 60 °C,
30 min). The samples were measured at angles of 0° and 80°.
Tabela 1: Koncentracija elementov na povr{ini PLLA. Vrednosti so
bile dolo~ene z metodo XPS na originalnih in z laserjem obdelanih
(9 mJ cm–2, 6000 pulzov) vzorcih, nato na vzorcih obdelanih z
laserjem, ki mu je sledilo `arjenje (9 mJ cm–2, 6000 pulzov, 60 °C, 30
min). Vzorci so bili merjeni pri kotih 0° in 80°.
Sample Angle (°) C (amountfractions, x/%)
O (amount
fractions, x/%)
Pristine
0 64.4 35.6
80 67.7 32.3
Laser
treatment
0 63.2 36.8
80 65.4 34.6
Laser
treatment +
annealing
0 62.6 37.4
80 67.7 32.3
The UV-Vis absorption spectra of the samples
exposed to laser fluences of 9 and 30 mJ cm–2 with 1000
and 6000 pulses and subsequently treated with thermal
Materiali in tehnologije / Materials and technology 50 (2016) 3, 331–335 333
I. MICHALJANI^OVA et al.: SURFACE PROPERTIES OF A LASER-TREATED BIOPOLYMER
Figure 1: Surface morphology of the samples treated with a combination of an excimer laser (6 mJ cm–2, 6.000 pulses) and thermal annealing
(60 °C, 30 min). The samples at the top were first annealed and then modified with the laser; the samples at the bottom were treated in the reverse
order. Ra represents the arithmetic mean surface roughness in nm.
Slika 1: Morfologija povr{ine vzorcev obdelanih s kombinacijo ekscimer laserja in `arjenja. Morfologija povr{ine vzorcev, obdelanih s
kombinacijo ekscimer laserja (6 mJ cm–2, 6,000 pulzov) in `arjenjem (60 °C, 30 min). Vzorci zgoraj so bili najprej `arjeni in nato modificirani z
laserjem, vzorci spodaj so bili obdelani v obratnem vrstnem redu. Ra predstavlja aritmeti~no sredino hrapavosti povr{ine v nm.
Figure 2: UV-Vis spectra of PLLA. UV-Vis spectra of: A) pristine
PLLA and samples exposed to laser fluence 9 mJ cm–2 and 30 mJ
cm–2 (1000 and 6000 pulses) and subsequently treated by thermal
annealing (60 °C, 30 min), B) 9 mJ cm–2, 1000 pulses + annealing, C)
9 mJ cm–2, 6000 pulses + annealing, D) 30 mJ cm–2, 1.000 pulses +
annealing and E) 30 mJ cm–2, 6.000 pulses + annealing.
Slika 2: UV-Vis spekter PLLA. UV-Vis spekter: A) prvoten PLLA in
vzorci izpostavljeni laserju pri 9 mJ cm–2 in 30 mJ cm–2 (1000 in 6000
pulzov) in nato obdelani z `arjenjem (60 °C, 30 min), B) 9 mJ cm–2,
1000 pulzov + `arjenje, C) 9 mJ cm–2, 6000 pulzov + `arjenje, D) 30
mJ cm–2, 1000 pulzov + `arjenje in E) 30 mJ cm–2, 6000 pulzov +
`arjenje.
annealing (60 °C, 30 min) are shown in Figure 2. In the
case of the spectra of the modified samples, there are
significant peaks at a position of approximately 275 nm.
The curve with the most significant peak represents the
treatment with a laser fluence of 9 mJ cm–2 and 6000 pul-
ses, belonging to a sample with an interesting spongious
morphology. The other modified samples also show at
least a "small" peak. After the combination of the laser
treatment and thermal annealing, a diagonal shift of the
absorbance curve was observed.
The cell adhesion represents the first stage of the
cell-substrate interaction, thus the quality of adhesion
influences cell ability to proliferate and differentiate in
the contact with a substrate. After successful adhesion,
the adaptation of the cells to the new environment (the
lag phase) occurs. For adhesion and proliferation studies
NIH 3T3 cells were chosen. The selected samples of the
modified PLLA substrate (9 or 30 mJ cm–2, 100 pulses,
annealing) were tested and compared with PLLA pristine
and control samples of polystyrene used for tissue cul-
tures (PS). Samples with 100 pulses were chosen, be-
cause with the increasing number of pulses the material
became brittle. From the Figure 3, it is apparent that the
best results were obtained on PS, which is commonly
considered as a model substrate. The PLLA pristine is a
material which biocompatibility can be improved by
plasma modification. It was shown that modified mate-
rial with 30 mJ cm–2 was slightly less cytocompatible,
but with 9 mJ cm–2 moderately improved its cytocompa-
tibility. Immediately after seeding cell adhesion was
found unaffected. In the Figure 4, there are introduced
selected pictures of cells growing on treated and pristine
PLLA samples.
4 DISCUSSION
By the combination of excimer laser and thermal
annealing, it is possible to prepare different surfaces of
PLLA with a wide range of surface roughness. The most
interesting part of this work is a wrinkle pattern creation.
Wrinkles could appear on polymer after annealing as a
thin bi-layer film (prepared by treatment and by anneali-
ng). In this case, the surface laser treatment and follow-
ing thermal annealing has created the wrinkle pattern.
We suggest that the structure is influenced also during
cooling. With less counts of pulses, the staminate
334 Materiali in tehnologije / Materials and technology 50 (2016) 3, 331–335
I. MICHALJANI^OVA et al.: SURFACE PROPERTIES OF A LASER-TREATED BIOPOLYMER
Figure 3: Tests of surface cytocompatibility. Dependence of the
number of adhered and proliferated NIH 3T3 cells (6, 24, and 72) h
after seeding on pristine PLLA (PLLA) and PLLA modified by laser
beam (9 mJ cm–2 or 30 mJ cm–2, 100 pulses) and subsequent thermal
annealing (60 °C, 30 min). The values for tissue polystyrene (PS) are
also shown for comparison.
Slika 3: Preizkus citokompatibilnosti povr{ine. Odvisnost {tevila
oprijetih in razmno`enih NIH 3T3 celic, (6, 24, 72) h po sejanju na
prvotni PLLA (PLLA) in na PLLA obdelan z laserskim `arkom (9 mJ
cm–2 ali 30 mJ cm–2, 100 pulzov), ki mu je sledilo `arjenje (60 °C, 30
min). Za primerjavo so prikazane tudi vrednosti za tkivni polistiren
(PS).
Figure 4: Mouse embryonic fibroblasts (NIH 3T3) growing on different substrates. Proliferated NIH 3T3 cells 72 h after seeding on various
substrates: A) modified PLLA (30 mJ cm–2, 100 pulses and subsequent thermal annealing (60 °C, 30 min); B) pristine PLLA, and C) tissue
polystyrene for comparison.
Slika 4: Mi{ji embrionski fibroblasti (NIH 3T3), ki so zrasli na razli~nih podlagah. Razmno`ene NIH 3T3 celice po 72 h po sejanju na razli~ne
podlage: A) modificiran PLLA (30 mJ cm–2, 100 pulzov in nato toplotna obdelava (60 °C, 30 min), B) prvotni PLLA in C) tkivni polistiren za
primerjavo.
patterns were built up. We propose that difference in the
structure was caused by the thickness of laser treated
layer, which was insufficient to create wrinkles. Instead
of that surface cracking occurred. We suggest that “rods”
on the PLLA surface were produced by crystallization of
the newly formed material from chopped polymer
strings. By reverse order of treatment, when the samples
were exposed to annealing and subsequently treated by
laser beam, the samples showed the same structure as the
samples treated just by laser without annealing. This
supports the theory, how the structure was formed.
The peak around area of 275 nm is typical for transi-
tions of n electrons to the * excited state, and represents
the presence of C=O group. This type of transition needs
an unsaturated group in the molecule to provide
the  electrons. The diagonal shift could be explained by
increasing concentration of double bonds.
Cytocompatibility tests show slight decrease of
ability to support cell proliferation for the PLLA treated
by high laser fluence, the biocompatibility increases for
lower laser fluence. The application of lower energy has
therefore similar effect as plasma treatment which we
studied in our previous experiments.
5 CONCLUSIONS
Various types of surface structures on modified sam-
ples of PLLA were produced by exposure of KrF
excimer laser beam and subsequent thermal annealing:
• by UV-Vis spectroscopy we observed new C=O
groups and creation of double bonds;
• by choosing optimal input parameters, it is possible
to prepare structures from porous and spongeous to
flat biopolymer surface with staminate structures;
• the roughness is significantly dependent on laser
treatment and annealing input values;
• low laser fluence has a positive effect on cytocom-
patibility, but high laser fluence loses this effect.
Acknowledgements
This work was supported by the GACR under project
13-06609S.
6 REFERENCES
1 R. M. Rasal, A. V. Janorkar, D. E. Hirt, Progress in Polymer Science,
3 (2010) 35, 38–356, doi:10.1016/j.progpolymsci.2009.12.003
2 B. L. Hancock-Hanser, A. Frey, M. S. Leslie, P. H. Dutton, F. I.
Archer, P. A. Morin, Targeted multiplex next-generation sequencing:
advances in techniques of mitochondrial and nuclear DNA
sequencing for population genomics, Molecular Ecology Resources,
2 (2013) 13, 254–268, doi:10.1111/1755-0998.12059
3 E.-K. Yeong, S.-H. Chen, Y.-B. Tang, The Treatment of Bone
Exposure in Burns by Using Artificial Dermis, Annals of Plastic
Surgery, 6 (2012) 69, 607–610, doi:10.1097/SAP.0b013e318273f845
4 R. Lecover, N. Williams, N. Markovic, D. H. Reich, D. Q. Naiman,
H. E. Katz, Next-Generation Polymer Solar Cell Materials: Designed
Control of Interfacial Variables Acs Nano, 4 (2012) 6, 2865–2870,
doi:10.1021/nn301140w
5 M. Bolle, S. Lazare, Large scale excimer laser production of sub-
micron periodic structures on polymer surfaces, Applied Surface
Science, 1–4 (1993) 69, 31–37, doi:10.1016/0169-4332(93)90478-T
6 C.-M. Chen, S. Yang, Wrinkling instabilities in polymer films and
their applications, Polymer International, 7 (2012) 61, 1041–1047,
doi:10.1002/pi.4223
7 H. S. Kim, A. J. Crosby, Solvent-Responsive Surface via Wrinkling
Instability, Advanced Materials, 36 (2011) 23, 4188–4192,
doi:10.1002/adma.201101477
8 D. Y. Khang, H. Q. Jiang, Y. Huang, J. A. Rogers, A Stretchable
Form of Single-Crystal Silicon for High-Performance Electronics on
Rubber Substrates, Science, 5758 (2006) 311, 208–212, doi:10.1126/
science.1121401
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