EFFECTS OF FUNCTIONAL GROUPS, BIOSIGNAL MOLECULES

AND NANOTOPOGRAPHY ON CELLULAR PROLIFERATION

H.T. ŞAŞMAZEL*, S. MANOLACHE,

M. GÜMÜŞDERELİOĞLU
Atılım University, Department of Materials Engineering,
Incek, Gölbaşı, Kizilcasar Mahallesi 06836, Ankara, Turkey

Abstract. The aim of this study is the development of novel cell support
materials for fibroblast cell cultivation by using low-pressure plasma
assisted treatment. Poly(ε-caprolactone) (PCL) membranes were prepared
by solvent-casting technique. The plasma assisted treatment was focused on
generating a nano-topography and obtaining COOH functionalities on the
surface of the membranes. The immobilization of biomolecules onto the
PCL membranes was realized after the plasma treatment. The membranes
prepared were characterized by various methods before and after the bio-
modification. L929 mouse fibroblasts were used for cell culture evaluation.
The prominent roles of surface nano-topography and carboxylic groups
generated by the plasma in obtaining better cell growth on PCL surfaces are
highlighted in this study.

Keywords: poly(ε-caprolactone); low pressure plasma; biomolecules immobilization;

cell proliferation

1. Introduction

Synthetic polymers need selective modifications in order to introduce spe-

cific functional groups (e.g. amines, carboxyls) to the surface for the bind-
ing of biomolecules and enhancing cell growth.1 Surface modification of
polymers can be achieved by wet (acid, alkali), dry (plasma) and radiation
treatments (ultraviolet radiation, laser) without affecting the bulk proper-
ties.2–4 Plasma treatments have many advantages compared to wet-chemical
methods.5
______
*
htsasmazel@atilim.edu.tr+

J.P. Reithmaier et al. (eds.), Nanostructured Materials for Advanced Technological Applications, 533
© Springer Science + Business Media B.V. 2009
534 H.T. ŞAŞMAZEL ET AL.

In the present study, utilizing these advantages, we introduced a new

plasma assisted treatment of PCL membranes in order to immobilize insulin
or heparin biomolecules through PEO (polyoxyethylene bis (amine)). The
biologically modified PCL membranes were tested with L929 mouse fibro-
blasts in cell culture experiments.

2. Experimental Section

2.1. PCL MEMBRANES

PCL (poly-ε-caprolactone) membranes were prepared by solvent-casting

technique and cut in the form of discs, with diameters of 12.5 mm, before
plasma treatment.

2.2. COLD PLASMA TREATMENT USING VACUUM ENVIRONMENT

Plasma assisted treatment was carried out in a cylindrical, capacitively

coupled RF plasma reactor equipped with a 40 kHz power supply in three
steps: H2O/O2 plasma treatment; in situ or ex situ gas/solid reaction (oxalyl
chloride vapors; pressure 65 Pa; time 30 min); and hydrolysis (open labora-
tory condition; 2 h) for final –COOH functionalities.

3. Characterization Studies

Experimental parameters such as the flow rates of water and oxygen, power,
pressure and time of the plasma modifications were optimized according to
a design of experiments (DoE) program (Design Expert 7, Stat-Ease, Inc.,
Minneapolis, MN). COOH and OH functionalities on the modified surfaces
were detected quantitatively by using fluorescent labeling technique and an
UVX 300G sensor. Structural chemical information of untreated and plasma
treated PCL membranes were acquired using pyrolysis gas chromatography/
mass spectroscopy (GC/MS) analysis. Electron spectroscopy for chemical
analysis (ESCA) analysis was used to evaluate the relative surface atomic
compositions and the carbon and oxygen linkages located in non-equivalent
atomic positions. Atomic force microscopy (AFM) analysis (Molecular
Imaging PicoSPM instrument; contact mode; silicon and hydrazine (HZD)
functionalized cantilevers) were carried out on H2O/O2 plasma treated, oxalyl
chloride functionalized and untreated PCL samples in order to observe the
micro- and/or nano-topography of surfaces.
 PLASMA NANOTOPOGRAPHY AND CELLULAR PROLIFERATION 535
4. Biological Modification of PCL Membranes

The biomodification of PCL membranes with insulin and heparin biosignal

molecules in the presence of a PEO spacer was realized after water/O2 plasma
treatment. The success of the immobilization process was checked qualita-
tively with ESCA. In addition, the amount of immobilized biomolecules
was determined by using fluorescent labeling techniques.

5. L929 Cell Culture

Anchorage-dependent L929 mouse fibroblasts [HUKUK (Cell Culture

Collection) 92123004] were cultured using Dulbecco’s Modified Eagle’s
Medium supplemented with 10% (v/v) fetal bovine serum in 24-well poly-
styrene tissue-culture dishes containing PCL membranes. Untreated and
plasma-oxalyl chloride treated discs were placed under UV light for 30 min
in order to sterilize them. Biomolecule-immobilized discs were sterilized by
UV light for 10 min. An MTT assay was used in order to determine the pro-
liferation of cells in the course of a 6-day cell culture period.

6. Results and Discussion

In our previous study,6 the glass transition temperature Tg, the melting
temperature Tm and the values of melting enthalpy ΔHm of PCL membranes
were determined by differential scanning calorimetry (DSC) analysis at –
59.20°C, 62.24°C and 68.00 Jg−1, respectively. The FTIR spectrum of a
PCL membrane exhibited the characteristic peaks of PCL structures in the
range of 400–4,000 cm−1.

6.1. PLASMA TREATMENT

After evaluation with DoE, the best conditions for plasma treatment of
further samples were selected as follows: flow rate = 5 sccm; pressure = 53
Pa; power = 35 W; time = 3.5 min. For these contiditions, the carboxyl
density is high without decreasing the OH density too much; they are
extremely close to some of the computed optimal conditions, and the values
are easier to be controlled during the experiments.
The amount of carbon dioxide released by pyrolysis from the samples
increases for oxalyl chloride functionalized samples proving the efficient
attachment of carboxylic functionalities to the PCL surfaces. Water/O2
536 H.T. ŞAŞMAZEL ET AL.

plasma treated samples show a sensible increase of the CO2 amount, which
proves a negligible decarboxylation and the generation of carbonyl and/or
carboxyl groups during the plasma treatment. ESCA results showed that
decarboxylation of PCL membranes during the plasma procedure is negli-
gible. However, free carboxyl and ester functionalities are overlapping in
high resolution C 1s peaks, so labeling was performed for identifying/quan-
tifying free carboxyls. High resolution AFM images (1 × 1 µm) revealed
that nano patterns were more affected than micro patterns by the plasma
treatments. AFM images recorded with HZD functionalized tips showed an
increased size of the features on the surface, which suggests a higher den-
sity of carboxyls on these nanotopographical elements (Figure 1).

Figure 1. AFM images of PCL membranes: A – untreated; B – water/oxygen plasma treated;

C – water/oxygen plasma treated and oxalyl chloride functionalized; D – same as C,
recorded with a hydrazine functionalized tip.
 PLASMA NANOTOPOGRAPHY AND CELLULAR PROLIFERATION 537
6.2. BIOMOLECULE IMMOBILIZATION

ESCA results proved the immobilization of biosignal molecules qualitatively.

By using fluorescent labeling techniques, the average amounts of immobi-
lized insulin and heparin on PCL surfaces were determined as 219.23 and
271.20 nmol/cm2, respectively.

6.3. L929 CELL CULTIVATION

According to the results of cell proliferation studies, heparin immobilized

PCL samples were the most suitable materials for L929 cell growth (Figure 2).

1,2

1
Absorbance (540 nm)

0,8 A
B
0,6 C
D
0,4 E

0,2

0
3 6
Time (Day)

Figure 2. L929 fibroblast cell growth on PCL membranes. A: Unmodified; B: Low pressure
water/O2 plasma treated and then oxalyl chloride functionalized; C: PEO grafted; D: Insulin
immobilized; E: Heparin immobilized.

7. Conclusion

The present study showed that low pressure water/O2 plasma assisted treat-
ment method works well for the immobilization of biomolecules onto PCL
membranes; these improved PCL membranes can be used as artificial tissue
538 H.T. ŞAŞMAZEL ET AL.

substituents. Our suggestion for related future studies is an investigation of

the effects of the nano-topography created by the plasma and the bio-
molecules on the cell growth in detail.

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