Biomaterials
Science
PAPER
Cite this: Biomater. Sci., 2013, 1, 850
Received 1st March 2013,
Accepted 4th April 2013
DOI: 10.1039/c3bm60055f
www.rsc.org/biomaterialsscience
Cell phenotypic changes of mouse connective tissue
fibroblasts (L-929) to poly(ethylene glycol)-based gels†
Christine Strehmel,
a
Zhenfang Zhang,
a
Nadine Strehmel*
b
and Marga C. Lensen*
a
Cellular responses to various gels fabricated by photoinitiated crosslinking using acrylated linear and
multi-arm poly(ethylene glycol) (PEG)-based and poly(propylene glycol)-b-poly(ethylene glycol) precur-
sors were investigated. While no protein adsorption and cell adhesion were observed on the hydrophilic
PEG-based gels, protein adsorption and cell adhesion did occur on the more hydrophobic gel generated
from the block copolymer precursor. Murine fibroblast viability on the poly(ethylene glycol)-based gels
was studied in the course of 72 h and the results indicated no cytotoxicity. In a systematic study, extra-
and intracellular metabolites of the murine fibroblasts cultured on these PEG-based gels were examined
by GC-MS. Distinct intra- and extracellular changes in primary metabolism, namely amino acid metab-
olism, glycolysis and fatty acid metabolism, were observed. Cells cultured on the polymeric gels induced
more intense intracellular changes in the metabolite profile by means of higher metabolite intensities
with time in comparison to cells cultured on the reference substrate (tissue culture polystyrene). In con-
trast, extracellular changes of metabolite intensities were comparable.
Introduction
Synthetic polymers, such as poly(ethylene glycol) (PEG), poly-
(2-hydroxyethyl methacrylate) (PHEMA) and poly(vinyl alcohol)
(PVA), have been frequently used in medicine and pharma-
cology for fabrication of various devices and for tissue engin-
eering applications due to their versatile chemical and
mechanical properties.
1
Particularly, polymer gel networks
generated from derivatives of PEG, namely hydrogels, have
been examined and used for drug delivery and tissue engineer-
ing based on their ability to imbibe a large amount of water
and their soft and rubbery consistency.
2–5
Besides these ben-
eficial properties, PEG-based gels are known to be nontoxic,
non-immunogenic and possess anti-fouling properties.
6
Due
to the unique ability of PEG to block serum protein adsorption
as well as the ability to prevent prokaryotic and eukaryotic cell
adhesion, PEG can be further used as a surface coating
material for the passivation of biomaterial surfaces.
7–9
The
non-fouling character of such coatings relies on steric repul-
sion effects between the protein and the hydrated polymer
chains at the surface as well as on parameters such as chain
length and surface density.
10
Further studies proved the pre-
vention of unspecific protein adsorption using an ultrathin
star-shaped PEG layer.
11
Surface wettability or hydrophilicity of the surface and the
resulting protein adsorption and cell adhesion onto PEG-
based surfaces can be affected by changing the ratio of PEG
and poly(propylene glycol) (PPG) block lengths in tri-block
copolymer systems PEG-b-PPG-b-PEG.
12,13
These tri-block
copolymers, namely Pluronics
®
, are used for drug delivery
applications and as emulsion stabilizers in pharmaceutical for-
mulations.
14
In this study, we report on the use of polymeric
gels that are cross-linked from such tri-block copolymer pre-
cursors bearing telechelic acrylate groups.
In order to use polymeric (hydro)gels in biomedical appli-
cations, intense nutrient transport to cells as well as free
diffusion of waste products are required.
15
In this context,
metabolomics is a valuable tool to investigate biochemical
changes in the surrounding medium and to characterize small
molecules.
A variety of different analytical techniques, e.g. nuclear
magnetic resonance (NMR) and mass spectrometry coupled to
chromatographic techniques such as gas chromatography (GC)
and liquid chromatography (LC), can be used to measure the
metabolic status of cells.
16
Among these, GC-MS is one of the
most common analytical techniques to analyze changes within
primary metabolism, e.g. low-molecular-weight compounds
†Electronic supplementary information (ESI) available: In the ESI fluorescence
micrographs of protein adsorption to pure PEG-gel and to the block copolymer
are shown and mass spectral data of identified compounds as well as the swel-
ling degree, bulk elasticity and surface roughness of the crosslinked PEG-gels are
listed. See DOI: 10.1039/c3bm60055f
a
Technische Universität Berlin, Department of Chemistry, Nanostrukturierte
Biomaterialien, Straße des 17. Juni 124, 10623 Berlin, Germany.
E-mail: [email protected]
b
Leibniz Institute of Plant Biochemistry, Department of Stress and Developmental
Biology, Weinberg 3, 06120 Halle (Saale), Germany.
E-mail: [email protected]
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such as amino acids, lipids and organic acids, since this tech-
nique offers a high separation efficiency, high chromato-
graphic resolution and is further a relatively low-cost
method.
17
So far, metabolomics has been a useful tool for analyzing
biofluids, for drug development and for functional genomics
of plants.
18–20
In the context of biomaterials research, metabolomics has
only been used to study cellular responses to smooth and pat-
terned titanium substrates.
21
Nevertheless, there are no
studies concerning global metabolic changes of cells on syn-
thetic gels such as PEG-based substrates. Thus, we performed
untargeted metabolite profiling experiments of murine fibro-
blasts (L-929) on selected PEG-based substrates and on tissue
culture polystyrene using gas chromatography coupled to mass
spectrometry (GC-MS) in order to define extracellular and
intracellular metabolite changes between the different sub-
strates particularly with regard to cell phenotype in response
to nutritional, toxicological and environmental (e.g. surface
chemistry) changes.
Since our PEG-based gel compositions have not been
reported in the literature yet, cytocompatibility, wettability,
plasma protein adsorption and cellular behavior on these sub-
strates were investigated as well.
Experimental section
Acrylation of PEG monomers with hydroxyl end groups
PEG diol (3400 Da) and PEG-b-PPG-b-PEG diol (4400 Da) were
purchased from Sigma-Aldrich and 8-arm PEG octaol (15 kDa)
from Jenkem Technology USA. Potassium carbonate (K
2
CO
3
),
sodium chloride (NaCl) and magnesium sulfate (MgSO
4
)as
well as dry dichloromethane (CH
2
Cl
2
), petroleum ether and
acryloyl chloride were obtained from Sigma-Aldrich. All sol-
vents were of analytical grade.
PEG-b-PPG-b-PEG diol (4400 Da) was functionalized to yield
the acrylated derivative 3BC as previously described else-
where.
22
Briefly, PEG-b-PPG-b-PEG diol and potassium carbon-
ate were mixed in dry dichloromethane under a nitrogen-
based atmosphere. After cooling the solution to 0 °C, acryloyl
chloride was added and the mixture stirred at 50 °C for 2 days.
Subsequently, the solution was filtered and poured into cold
petroleum ether. Following stirring for 10 min, the petroleum
ether was decanted. Finally, the crude product was dissolved
in dichloromethane and washed with saturated sodium
chloride solution. The organic layer was collected and dried
with magnesium sulfate overnight. After filtration, the solvent
was removed under reduced pressure resulting in a colourless
liquid with a moderate yield (64%).
1
H NMR (CDCl
3
)of3BC:
OCH
2
CHCH
3
O 1.12 ppm; OCH
2
CHCH
3
O 3.38 ppm; OCH
2
CH-
CH
3
O 3.52 ppm; OCH
2
CH
2
O 3.63 ppm; (CvO)OCH
2
4.30 ppm;
vC–Htrans 5.83 ppm; CHvC 6.15 ppm; vC–Hcis 6.42 ppm.
The acrylation of PEG diol (3400 Da) and 8-arm PEG octaol
(15 kDa) was conducted as outlined above for PEG-b-PPG-b-
PEG diol (4400 Da) to yield the new acrylated PEG and 8-PEG
derivatives. In these cases, the mixtures were stirred at 60 °C
for 4 days. A white solid was obtained for PEG and 8-PEG with
a yield of 70% and 72%, respectively. NMR analysis revealed
complete conversion of hydroxyl-groups into acrylate func-
tions.
1
H NMR (CDCl
3
)ofPEG:OCH
2
CH
2
O 3.64 ppm; (CvO)-
OCH
2
4.31 ppm; vC–Htrans 5.83 ppm; CHvC 6.15 ppm;
vC–Hcis 6.42 ppm.
1
H NMR (CDCl
3
)of8-PEG:OCH
2
CH
2
O
3.64 ppm; (CvO)OCH
2
4.31 ppm; vC–Htrans 5.83 ppm;
CHvC 6.15 ppm; vC–Hcis 6.42 ppm.
Physicochemical properties of PEG-based precursors are
shown in Table 1.
Gel formation
The liquid, acrylated precursor PEG-b-PPG-b-PEG (3BC)was
mixed with 1% of the photoinitiator (PI) Irgacure 2959 (Sigma-
Aldrich) in acetone. Subsequently, a mild stream of nitrogen
was applied to evaporate the solvent. In order to mix the PI
with the solid precursors, i.e. linear PEG (PEG) and 8-arm PEG
(8-PEG), aqueous solutions (33 wt%) containing 1% of PI (1 wt%
with respect to the amount of the precursor) were prepared.
For surface analysis, the solid precursors PEG and 8-PEG were
transformed into a melt using an oven.
Silicon wafers were cleaned with acetone, activated using
hydrogen peroxide–sulphuric acid (3 : 7, v/v) and fluorinated
with trichloro(1H,1H,2H,2H-perfluorooctyl)silane (Sigma-
Aldrich) prior to use. Subsequently, 50 μl of the selected PEG
precursor mixtures were dispensed on fluorinated silicon
wafers (CrysTec GmbH), capped with a cover glass (18 mm ×
18 mm; Carl Roth GmbH & Co. KG) and exposed to UV light
(λ= 365 nm, Vilber Lourmat GmbH) for 30 min using a working
distance of 10 cm, in a nitrogen-filled glovebox. Finally, the
cured transparent gels were peeled offwith tweezers and kept
in clean petri dishes (VWR International GmbH) until further
use.
Swelling tests
After polymerization, gels were washed in deionised water to
remove unreacted precursors and dried in a vacuum oven for
48 h. Subsequently, the dry gels of known weight (W
d
) were
immersed in a cell culture medium at 37 °C. After appropriate
time points, swollen gels were taken out from the medium,
blotted dry with a tissue paper and weighed again (W
s
)
Table 1 Physicochemical properties of PEG-based precursors: PEG diacrylate
(PEG), PEG-b-PPG-b-PEG (3BC) and 8-arm PEG acrylate (8-PEG). Values were
obtained from the manufacturer. R: hexaglycerin core structure, r.t.: room
temperature
Material PEG diacrylate
(PEG)PEG-b-PPG-b-PEG
diacrylate (3BC)8-arm PEG
acrylate (8-PEG)
Structure
M
w
[kDa] 3.4 4.4 15
Chain length n∼72 n+p∼12; m∼57 n∼40
PEG [%] 100 ∼30, (∼70% PPG) 100
State at r.t. Solid Liquid Solid
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immediately. The swelling degree (SD) was determined accord-
ing to eqn (1), where W
s
is the gel mass after swelling and W
d
is the dry gel mass.
22
SD½%¼WsWd
Wd100 ð1Þ
Surface analysis
Water contact angles were measured by the sessile drop
method using a goniometer (Data Physics) equipped with a
video camera. Two μl of deionised water were deposited onto
the material surfaces using the goniometer syringe. Images of
droplets were taken at 21 °C. Contact angle values were
recorded and averaged from five independent samples.
Protein adsorption
In water swollen PEG-based gels PEG,3BC and 8-PEG (1 cm ×
1 cm) were placed in μ-slides (Ibidi GmbH) and incubated with
0.3 ml of a solution of fluorescently labelled albumin (50 μg
ml
−1
fluorescein isothiocyanate conjugated albumin in phos-
phate buffered saline, Sigma-Aldrich) for 1 h in a humidified
and thermostated incubator (37 °C and 5% of CO
2
). After the
incubation period, samples were washed twice with phosphate
buffered saline (PBS) and analysed by fluorescence microscopy
using an inverted microscope (Axio Observer.Z1, Carl Zeiss)
equipped with a light emitting diode source (Colibri, Carl
Zeiss). Pictures were analysed using the Axio Vision software (V
4.8.2, Carl Zeiss).
Cell culture
Mouse connective tissue fibroblasts (L-929) were grown in
75 cm
2
cell culture flasks (Greiner Bio-One) in a RPMI
1640 medium containing 10% fetal bovine serum and 1%
penicillin/streptomycin (all PAA Laboratories GmbH). The cul-
tures were incubated in a humidified incubator (CB 150,
Binder GmbH) at 37 °C and 5% CO
2
atmosphere as previously
described elsewhere.
22
For the following experiments L-929
cells were used between passages 9 and 15. The cell culture
medium was refreshed every second day.
Cell viability
Live/dead assay. PEG-based gels (1 cm × 1 cm) were washed
with ethanol (70%), rinsed in Dulbecco’s PBS (DPBS, PAA Lab-
oratories GmbH) and placed in a μ-slide (Ibidi GmbH). 300 μl
of a cell suspension containing 50 000 cells were seeded into
each well and incubated at 37 °C, 5% CO
2
atmosphere and
100% humidity. The viability of cells on the three gels PEG,
3BC, and 8-PEG was estimated after 24 h, 48 h and 72 h of
incubation. Following incubation, cells were stained with
100 μl of a vitality staining solution containing fluorescein di-
acetate (stock solution 0.5 mg ml
−1
in acetone, Sigma-Aldrich)
and propidium iodide (stock solution 0.5 mg ml
−1
in Ringer’s
solution, Fluka). Viable and dead cells were quantified by fluo-
rescence microscopy.
The cell culture medium was not exchanged during the
examined period due to the fact that particularly cells seeded
on PEG or 8-PEG do not adhere to the polymer surface and
would be sucked away. The number of viable and dead cells
was counted; values were determined in triplicate.
Water soluble tetrazolium assay (WST). PEG-based gels
(0.5 cm in diameter) were washed with ethanol (70%), rinsed
in DPBS and placed in 96-microwell plates (Greiner Bio-One).
For the indirect experiment, 100 μl of the cell culture medium
were placed on top of each gel and incubated for 24 h, 48 h
and accordingly 72 h at 37 °C and 5% CO
2
.
5000 L-929 cells per 100 μl were seeded into each well of a
96-microwell plate. The plate was pre-incubated for 24 h at
37 °C and 5% CO
2
. Next, the cell culture medium was aspi-
rated through a pipette. 100 μl of the medium that had been
on top of the gels for 24 h, 48 h or 72 h were added on top of
the newly seeded cells. After an incubation period of 24 h,
10 μl of Cell Counting Kit-8 (Sigma-Aldrich) containing the
water soluble WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitro-
phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium
salt] were added to each well. The 96-microwell plate was incu-
bated for 1 h in an incubator and the absorbance of the forma-
zan product (λ
450 nm
) was measured using a microplate reader
(infinite
®
200, Tecan). Wells containing medium without cells
were used as background control. The number of viable cells
was expressed as the relative percentage of the control; values
were determined in triplicate. Cells cultured on tissue culture
polystyrene (TCPS) were used as control.
Optical and scanning electron microscopy
300 μl medium containing 50 000 cells were seeded onto every
material. After 24 h, 48 h and 72 h optical images were taken
with the Axio Oberserver.Z1 (Carl Zeiss). The diameter of cells
on PEG,3BC,8-PEG and on tissue culture polystyrene (TCPS)
was measured using the Axio Vision software (V4.8.2 Carl
Zeiss).
For scanning electron microscopy (SEM) of L-929 cells on
3BC, cells were rinsed with PBS and fixed with formaldehyde
(4%, Carl Roth GmbH & Co. KG) for 30 min. Subsequently, the
samples were dehydrated in a graded acetone series, dried
with critical point drying (CPD 030, Baltec) as previously
described and sputtered with gold using a sputter coater (SCD
030, Balzers).
23
Scanning electron images were taken with a
Hitachi S-520 using an acceleration voltage of 20 kV and a
working distance of 10 mm. Pictures were taken using the
Digital Image Processing System (2.6.20.1, Point Electronic).
Since cell adhesion on the pure PEG-based gels PEG and
8-PEG was minimal, hardly any cells would withstand the
rinsing and fixation procedure and therefore these samples
were not investigated by SEM.
Metabolite profiling via GC-MS
Collection of intra- and extracellular metabolites. The PEG-
based gels PEG,3BC and 8-PEG (1.4 cm in diameter) were
washed with ethanol (70%), rinsed in DPBS and placed in a
24-microwell plate (Becton Dickinson). L-929 cells (50 000 cells
per ml) were seeded onto each sample and cultured for a
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defined time period (24–72 h) in a humidified incubator with
5% CO
2
and 37 °C. TCPS was used as reference material.
In the case of the reference material TCPS and the PEG-
based gel 3BC, on which cells could adhere, the supernatant
was removed and stored at −80 °C until further analysis. The
adherent cells were washed with DPBS to remove extracellular
metabolites and treated with Trypsin-EDTA (PAA Laboratories
GmbH) to detach the cells from the polymer surface. Sub-
sequently, detached cells were resuspended in a RPMI 1640
cell culture medium. The solution was transferred into a
falcon tube (VWR International GmbH) and centrifuged at
1300 rpm for 5 min at 4 °C (Eppendorf 5810R). Finally, the cell
pellet was washed twice with DPBS. Cells were counted with a
haemocytometer (Paul Marienfeld GmbH & Co. KG). Cells
from three wells were combined into one Eppendorf-tube
(aliquot approx. 1 × 10
6
cells). The cell pellet was immediately
frozen in liquid nitrogen after removing DPBS and stored at
−80 °C until further analysis. Experiments were performed in
triplicate.
The cell culture medium along with the non-adherent cells
on PEG and 8-PEG was collected and centrifuged after 24 h,
48 h and 72 h, respectively. The supernatant was stored at
−80 °C until further use, while the cell pellet was washed twice
with DPBS. Cells were counted according to the procedure
described for 3BC and TCPS. The main preparation steps for
the GC-MS based analysis of extracellular and intracellular
metabolites are illustrated in Fig. 1.
Extraction of intracellular metabolites. Metabolite extrac-
tion from cells was conducted as previously described.
24
Briefly, 400 μl of ice-cold extraction solvent, i.e. methanol–
chloroform–water (1 : 1 : 0.1, v/v/v), were added to the frozen
cell pellet. Subsequently, the mixture was vortexed, thawed in
an ice bath for 10 min and transferred to liquid nitrogen for
an additional 10 min. The thawing and freezing were repeated
twice. After centrifugation (5 min at 4 °C and with 1350 rpm),
the supernatant was transferred into a new Eppendorf-tube.
The cell pellet was re-extracted with 400 μl of ice-cold extrac-
tion solvent, treated at 70 °C for 15 min and centrifuged.
Prior to derivatisation, 800 μl of the cell pellet extract con-
taining intracellular metabolites were evaporated to dryness in
a speed-vac concentrator (RVC 2-25CD plis, CHRIST).
Derivatisation of intra- and extracellular metabolites. For
derivatisation of intracellular metabolites, 40 μl of methoxy-
amine hydrochloride in pyridine (20 mg ml
−1
, Sigma-Aldrich)
were added, the mixture was vortexed (Vortex Genie 2, Scienti-
fic Industries) and incubated at 40 °C (Thermo comfort 2 ml,
Eppendorf) for 1.5 h. Following incubation, 80 μl of an alkane
mixture containing N,O-bis(trimethylsilyl)trifluoroacetamide
(BSTFA, Macherey–Nagel GmbH & Co. KG) and alkanes
(dodecane, pentadecane, nonadecane, docosane, octacosane,
dotriacontane; 0.22 mg ml
−1
in pyridine) were added and incu-
bated at 40 °C for 30 min. The mixture was centrifuged at
14 000 rpm for 1 min (Eppendorf 5415R) and the supernatant
transferred into a glass vial (Chromatographiezubehör Trott).
Extracellular metabolites were derivatised using the MPS
2XL-Twister Autosampler (Gerstel GmbH & Co. KG). Prior to
derivatisation, 10 μl medium were evaporated until dryness in
a speed-vac concentrator (RVC 2-25CD plis, CHRIST). 20 μlof
methoxyamine hydrochloride in pyridine (20 mg ml
−1
), 40 μl
of BSTFA and 5 μl of the alkane mixture were used. All other
conditions were maintained as described for the derivatisation
of intracellular metabolites.
GC-MS analysis. GC-MS analysis was performed on a 6890
GC System (Agilent Technologies) hyphenated to a 5975 QUAD
Fig. 1 Schematic illustration of the main preparation steps for the analysis of intracellular (cell pellet; depicted in grey) and extracellular (medium; depicted in
black) metabolites. Methoxyamine (MeOX) hydrochloride in pyridine and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) were used for derivatization.
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Detector (Agilent Technologies). One μl derivatised sample was
injected splitless at 230 °C with a purge flow of 20 ml min
−1
,
while the purge was turned on after 1 min. Analytes were sep-
arated on a ZB-5MS column (30 m in length; 0.25 μm film
thickness; 0.25 mm inner diameter and 10 m EZ-guard pre-
column) with a flow rate of 1 ml min
−1
(Phenomenex). The
initial oven temperature was set at 70 °C and raised after
1 min to 300 °C at 9 °C min
−1
. This final temperature was
maintained for 5 min and then the GC adjusted to its initial
conditions before the next injection was started. Helium was
used as a carrier gas and operated at constant flow (1 ml
min
−1
). The transfer line was set at 300 °C. The QUAD-detec-
tion was operated with 3 scans per second in the range of
70–600 amu.
Data analysis and statistics
Data analysis was performed with the help of the MetAlign
software (version 10/2007) and the TagFinder software (version
4.1), in succession.
25
In short, peak intensities above 1000
arbitrary ion current units were imported, aligned and
grouped according to their common retention time and mass
spectral features. The relevant mass spectral features were
identified with the help of the Golm Metabolome Database
(gmd.mpimp-golm.mpg.de; version June 2011).
For statistics, peak intensities were baseline-corrected and
normalized to cell number and abundance of the alkanes;
medium samples were only normalized to alkanes. Then, the
data were mean-centred per analyte, log 2-transformed and
submitted to statistical analysis (two-way analysis of variance,
ANOVA: time × material). This was conducted with the help of
the Multi Experiment Viewer software (MeV, version 4.7.2).
26
Results and discussion
Surface analysis, protein adsorption and cell morphology
PEG-based gels with different swelling properties were fabri-
cated by photoinitiated crosslinking using acrylated linear and
multi-arm PEG-based precursors as well as poly(propylene
glycol)-b-poly(ethylene glycol) precursors. The crosslinked gels
prepared with PEG and 8-PEG can take up more water than the
crosslinked gel prepared with 3BC.
22
The swelling degree of
the gels after 24 h is shown in the ESI (Table S3†).
The cellular response and protein adsorption of L-929 cells
to the materials PEG,3BC,8-PEG and TCPS were investigated
(using optical-, scanning electron- and accordingly fluo-
rescence microscopy) and correlated to their wettability, since
cell adhesion and protein adsorption onto biomaterial sur-
faces are known to be influenced amongst others by the wett-
ability of the surface.
27
An optimum in protein adsorption and
cell adhesion occurs on surfaces with an intermediate wettabi-
lity ranging from 60 to 80°.
28
Consequently, too hydrophilic
(water contact angle below 60°) and too hydrophobic sub-
strates (water contact angle above 80°) support protein adsorp-
tion and cell adhesion to a lesser extent.
According to our expectation due to the hydrophilic surface,
smooth PEG and 8-PEG were anti-adhesive (Fig. 2A), showing a
round-shaped cell morphology (17 ± 1 μm in diameter) indica-
tive of the absence of spreading, and cell clustering, Fig. 2B.
Furthermore, no protein adsorption was observed on the
hydrophilic gels PEG and 8-PEG as expected, Fig. S1.†In con-
trast, the more hydrophobic substrates TCPS and 3BC did
induce protein adsorption, cell adhesion and spreading,
resulting in a cell diameter of 63 ± 15 μm and 57 ± 15 μm,
respectively.
In fact, PEG surfaces are very well known to prevent protein
adsorption and cell adhesion.
29
Recently, we have demon-
strated the cell-repellent effect towards a hydrogel prepared
from a star-shaped PEG with acrylate end groups due to its
hydrophilic surface (static contact angle 43° ± 1°).
30
A likewise,
relatively low contact angle (52°) was detected for a gel pre-
pared from a linear PEG macromolecule with diacrylate end-
groups.
31
Changing the ratio of PEG in tri-block copolymer systems
increases the hydrophobicity of the surface, and thereby
enables more protein adsorption and cell adhesion.
32
Thus,
the surface of 3BC was indeed found to be more hydrophobic
in comparison to PEG and 8-PEG, since the gel prepared from
3BC contains a small PEG (30%) and a large PPG (70%)
content in this gel formulation.
Cell viability
For the prospective use of PEG-based gels in biomedical appli-
cations, gels require excellent cytocompatibility. Nevertheless,
unreacted monomers and photoinitiator fragments can be
physically trapped in UV-cured gels. These compounds can be
released into the cell culture medium, most likely leading to
in vitro toxicity, which can result in cell death, loss of membrane
integrity, altered cell morphology and reduced biosynthetic
activity.
33
Thus, cytocompatibility of PEG,3BC and 8-PEG was
verified after direct cell contact using a live–dead assay and
Fig. 2 (A) Representative microscopy images showing the morphology of
L-929 cells on PEG,3BC and 8-PEG. (B) Contact angles onto various materials
were measured with the help of the sessile drop method. Cell diameter of L-929
cells on PEG,3BC,8-PEG and TCPS was obtained by the Axio Vision software.
The cell diameter of cells cultured on 3BC and TCPS was measured using the
long axis of the cells, while the cell diameter of cells cultured on PEG and 8-PEG
was obtained by measuring the cross-section dimension.
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indirectly by a water soluble tetrazolium salt based assay after
24 h, 48 h and 72 h of incubation.
The live–dead assay stains viable and non-viable cells by
means of fluorescein diacetate and propidium iodide, respecti-
vely. Viable cells express green fluorescence under UV illumi-
nation, representing transformation of fluorescein diacetate
into fluorescein by the action of esterases inside the living
cells. Propidium iodide passes through damaged plasma
membranes and binds to nucleic acids resulting in red fluo-
rescence.
34
Our live–dead staining results indicate that cells
remain viable after 24 h, 48 h as well as after 72 h (cell viability
is as follows [%]: PEG 98.5 ± 1.3, 97.7 ± 3.3, 98.1 ± 2.7; 3BC
98.9 ± 1.7, 97.9 ± 1.2, 97.4 ± 0.4 and 8-PEG 98.6 ± 2.5, 98.9 ±
1.9, 97.9 ± 3.6), Fig. 3A. Thus, loss of membrane integrity of
L-929 cells on PEG,3BC and 8-PEG did not occur within the
time frame of 72 h.
In a second step, we quantified the number of viable cells
via a nonradioactive colorimetric assay. In the course of this
assay, a tetrazolium salt is converted by a mitochondrial succi-
nate dehydrogenase into a yellow-coloured soluble formazan.
The cell viability of cells incubated with the medium that had
been on top of the PEG-based gels was not significantly
different from those cultured on the reference material TCPS,
Fig. 3B. As expected, L-929 cells showed more than 90% viabi-
lity after 24 h, 48 h and 72 h, respectively.
Thus, the direct and indirect contact of the PEG-based gels
(PEG,3BC and 8-PEG) with L-929 cells indicated no in vitro
cytotoxicity. Any remaining toxic components on the gel sur-
faces such as photoinitiator fragments and unreacted mono-
mers were most likely washed away with ethanol and
Dulbecco’s PBS before cell contact. The non-toxic potential of
our PEG-based gels corresponds to results obtained for UV-
curable gels prepared from linear PEG with diacrylate end
groups reported in the literature and star-shaped PEG precur-
sors investigated by our group.
35,36
Both groups report high
levels of cell viability by live–dead assay after an incubation
period of 24 h to 72 h.
Metabolite profiling via GC-MS
Next, we performed untargeted metabolite profiling exper-
iments of murine fibroblasts on smooth PEG-based gels PEG,
3BC and 8-PEG using gas chromatography coupled to mass
spectrometry to examine global intra- and extracellular meta-
bolic responses of L-929 cells on distinct substrates at three
default time points (24, 48 and 72 h). TCPS was used as refer-
ence material. During these studies we identified 16 extracellu-
lar metabolites and 15 intracellular metabolites changing in
the course of the predefined time frame in at least one of the
materials using two-way analysis of variance (two-way ANOVA).
Extracellular metabolites. Extracellular metabolites chan-
ging within time belong to diverse biochemical classes such as
amino acids (asparagine, cystine, glutamic acid, isoleucine,
leucine, methionine, ornithine, phenylalanine, proline, hydro-
xyproline, serine, threonine, tryptophan and tyrosine), organic
acids (lactic acid) and amides (urea), Fig. 4 and Table S2.†
The analysed medium of cells cultured on PEG,3BC and
8-PEG exhibited similar metabolic responses in comparison to
the medium of cells cultured on TCPS. Consequently, these
studies provide valuable information on the overall physiologi-
cal state of the cells.
As can be seen in Fig. 4, cells incubated on materials pre-
pared with precursor PEG,3BC,or8-PEG as well as cells on
TCPS released lactic acid continuously into the cell culture
medium. This corresponds to previous studies reported in the
literature, in which the release of metabolites by L-929 mouse
fibroblasts cultured on polystyrene tissue culture flasks into
the cell culture medium was determined.
37
Furthermore, an
incorporation of
13
C labelled carbon indicated that lactic acid
was derived from glucose. Since a high amount of glucose
(2000 mg l
−1
) is supplemented in our cell culture medium as
well, L-929 cells can utilize glucose as an energy source for cel-
lular maintenance and cellular growth.
In contrast, the amino acids isoleucine, leucine, methion-
ine, phenylalanine, threonine and tryptophan decreased
gradually in the course of the examined period in all cases.
These amino acids are supplied among others in the cell
culture medium, since they cannot be synthesized de novo by
eukaryotic cells.
38
The consumption of essential amino acids
by L-929 fibroblasts is consistent with a previous study reveal-
ing that the essential amino acids isoleucine, leucine, methio-
nine, phenylalanine, threonine and tryptophan are required
for cell metabolism.
39,40
Furthermore, these amino acids were
proven to be essential for cell growth in tissue culture.
41
A similar trend was observed for the non-essential amino
acids proline, hydroxyproline, serine and tyrosine. Interest-
ingly, asparagine, cystine, glutamic acid and ornithine
Fig. 3 (A) Live–dead assay indicating viable (green) and dead (red) L-929 cells
on 8-PEG after 24 h and 72 h. Dead cells are encircled. (B) WST assay illustrating
the cell viability of L-929 cells which were incubated with cell culture medium
that had been on top of PEG,3BC and 8-PEG for 24 h (white bar), 48 h (grey
bar) and 72 h (black bar).
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increased gradually in the medium for cells cultured on PEG,
3BC and 8-PEG, whereas, surprisingly, the medium of cells cul-
tured on TCPS exhibited a slight decrease of asparagine,
cystine and ornithine.
The increasing utilization of cystine, proline and tyrosine as
well as the increase of glutamic acid in the cell culture
medium has been described for L-929 fibroblasts in chemically
defined media.
37,39
The accumulation of glutamic acid in the
cell culture medium was expected, since glutamic acid is
derived from glutamine, which is a component in our cell
culture medium.
42
High levels of urea were obtained in media of cells cultured
on any material, while no clear temporal difference was
observed. The secretion of urea into the cell culture media
with distinct kinetics is described in the literature, indicating
a slight increase during 48 h.
43
Intracellular metabolites. A mixture of chloroform, metha-
nol and water is applicable for the extraction of intracellular
metabolites, and was used to recover the maximum amount of
metabolites.
24
As a result, both polar and lipophilic meta-
bolites are extracted.
GC-MS based metabolite profiling led to the identification
of 15 analytes belonging to diverse biochemical classes such
as amino acids (aspartic acid, glutamic acid, glycine and tyro-
sine), fatty acids (arachidic acid, myristic acid, palmitic acid,
stearic acid), monosaccharides (fructose and glucose 6-phos-
phate), nucleotides (adenosine monophosphate (AMP)), poly-
amines (putrescine), polyols (glucitol, myo-inositol), and
steroids (cholesterol), Fig. 5 and Table S1.†
The temporal metabolite profile of cells cultured on PEG,
3BC and 8-PEG was similar to the metabolite profile of cells
cultured on TCPS, although higher metabolic activity was
observed for cells cultured on PEG,3BC and 8-PEG when com-
pared to cells cultured on TCPS. This observation is surprising,
since the cell phenotype of non-adherent cells on PEG and
8-PEG reflects a similar metabolic activity in comparison to
adherent and spread cells on 3BC. Presumably, the higher
metabolic activity of cells cultured on PEG-based gels is
Fig. 4 Relative changes of ANOVA positive extracellular analytes. L-929 cells were incubated with PEG,3BC and 8-PEG for 24, 48 and 72 h. TCPS was used as refer-
ence material. The number of derivatised functional groups is given in brackets (TMS: trimethylsilyl). ANOVA positive analytes were maximum normalized.
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attributed to the more efficient nutrient exchange, by reason
that our PEG-based gels swell in the presence of water and the
cell culture medium, respectively.
A number of amino acids (i.e. aspartic acid, glutamic acid,
glycine and tyrosine) increased gradually in cells cultured on
PEG,3BC and 8-PEG, while only a moderate increase of amino
acid intensities within time was observed for cells cultured on
TCPS.
The increasing level of glycine and tyrosine in the cell pellet
material might have resulted from the consumption of extra-
cellular serine and phenylalanine, respectively. The corres-
ponding, gradual decrease of these amino acids is depicted in
Fig. 4. Moreover, accumulated aspartate and glutamate within
time can arise from enhanced tricarboxylic acid cycle (TCA)
activity, since aspartate and glutamate are derived from the
TCA cycle intermediates oxaloacetate and glutamine,
respectively.
Similar results concerning the accumulation of aspartate,
glycine, glutamate and tyrosine had been observed for
mesenchymal stem cells (MSC) cultured for 7 days on various
titanium substrates, using liquid chromatography tandem
mass spectrometry.
21
An increased capacity for protein syn-
thesis on the titanium substrates was proposed as explanation
for the accumulation of amino acids. Concerning our
materials, cells cultured on PEG-based gels (PEG,3BC and
8-PEG) possibly increased their capacity for protein synthesis
as well.
Amino acids with nitrogen-containing residues can break
down into polyamines like putrescine. A cumulative response
within time was observed for cells cultured on PEG,3BC and
8-PEG. A slight increase was also observed for cells cultured on
TCPS. Since the cell culture medium was not exchanged at all
during the examined period due to non-adherent cells on PEG
and 8-PEG, an accumulation of putrescine within time is
plausible. However, putrescine is toxic and can inhibit cell pro-
liferation even at low concentration.
44
Nevertheless, direct and
indirect cell contact with PEG,3BC,8-PEG and TCPS indicated
no toxicity over the examined period.
Fig. 5 Relative amount of ANOVA positive intracellular analytes. L-929 cells were cultured on PEG,3BC and 8-PEG for 24, 48 and 72 h. TCPS was used as reference
material. The number of derivatised functional groups is given in brackets (TMS: trimethylsilyl, MeOX: methoxyaminated). ANOVA positive analytes were maximum
normalized.
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Besides the temporal increase of amino acids and polya-
mines, elevated monosaccharide (fructose, glucose 6-phos-
phate) and polyol (glucitol, myo-inositol) abundances were
observed for cells cultured on PEG,3BC and 8-PEG, while
these metabolites were only slightly increased with respect to
cells cultured on TCPS. Elevated intracellular fructose and glu-
citol had been observed for Chinese hamster ovary (CHO)
cells, arising from glucitol pathway, an alternate pathway for
glucose.
45
Myo-inositol derives from glucose 6-phosphate,
which is an intermediate in glycolysis. Interestingly, both ana-
lytes accumulate during the examined period.
The relative intensity of adenosine monophosphate (AMP)
is gradually increased for cells cultured on PEG,3BC and
8-PEG as well as slightly enhanced for cells cultured on TCPS.
This could be attributed to the labile adenosine triphosphate
(ATP), which is an important energy transfer compound within
cells. Due to the high reactivity of ATP, we could only detect
the breakdown product AMP after derivatisation.
46
The cell membrane of eukaryotic cells consists of phospho-
lipids and cholesterol, among others, while fatty acids are part
of phospholipids. Relative cholesterol and fatty acid (e.g. ara-
chidic acid, myristic acid, palmitic acid and stearic acid) levels
increased within time independent of the material. Since
cholesterol is an essential structural component for cell mem-
branes and is further required to ensure membrane stability,
permeability and fluidity, spread cells cultured on 3BC and
TCPS were expected to exhibit a higher relative cholesterol
level in comparison to round cells cultured on PEG and
8-PEG.
47
Interestingly, an intense cholesterol accumulation was
observed for round cells cultured on PEG and 8-PEG as well as
for spread cells cultured on 3BC in comparison to spread cells
cultured on TCPS.
Concerning fatty acid accumulation, an elevated phospholi-
pid breakdown resulting in the build-up of fatty acids under
conditions of oxygen deficiency has been reported.
48
Since
oxygen levels in cell cultures are cell density dependent, an
increase of fatty acids, as our results show, is
comprehensible.
49
Conclusions
In conclusion, PEG and 8-PEG containing pure poly(ethylene
glycol) are protein and cell repellent due to the hydrophilic
surface, while protein adsorption and cell adhesion occur on
the more hydrophobic 3BC surface containing a relatively
small PEG content. Thus, protein adsorption and cell adhesion
can be consequently influenced by changing the ratio of cell
repellent material, namely PEG. Our results also indicated that
all the studied PEG-based gels are cytocompatible.
Our investigations concerning metabolism reveal that
GC-MS analysis can be used to evaluate cell responses to bio-
material surfaces. Indeed, our untargeted metabolite profiling
results are not coincident with cell phenotypes. In addition,
untargeted metabolite profiling revealed similar trends of
extracellular metabolites obtained from the medium of cells
cultured on PEG,3BC,8-PEG and TCPS, respectively. In con-
trast, relative intensities of intracellular metabolites obtained
from round L-929 cells on PEG and 8-PEG as well as spread
cells on 3BC differed from those on TCPS. This leads to the
conclusion that cells cultured on PEG-based gels have an
increased intracellular metabolic activity in comparison to
cells cultured on TCPS. In the case of PEG,3BC and 8-PEG,
which swell in the presence of water or cell culture medium, a
better diffusion of nutrients and an eventually increased
metabolism can be held responsible for this observation.
Acknowledgements
The authors thank Dr J. Lehmann, Fraunhofer Institute for
Cell Therapy and Immunology Leipzig, for kindly providing
mouse connective tissue fibroblasts (L-929) and greatly
acknowledge funding in the form of a Sofja Kovalevskaja
Award granted to M. C. Lensen by the Alexander von Humboldt
Foundation and funded by the Federal Ministry for Education
and Research (BMBF). The authors also thank the Deutsche
Forschungsgemeinschaft (DFG) for financial support within
the framework of the German Initiative for Excellence the
Cluster of Excellence “Unifying Concepts in Catalysis”(EXC
314) coordinated by the Technische Universität Berlin.
References
1 E. S. Place, J. H. George, C. K. Williams and M. M. Stevens,
Chem. Soc. Rev., 2009, 38, 1139–1151.
2 N. B. Graham and M. E. McNeill, Biomaterials, 1984, 5,27–
36.
3 J. K. Tessmar and A. M. Göpferich, Macromol. Biosci., 2007,
7,23–39.
4 H. Park and K. Park, in Hydrogels and Biodegradable Poly-
mers for Bioapplications, ed. R. M. Ottenbrite, S. J. Huang
and K. Park, American Chemical Society, Washington DC,
1996, p. 2.
5 K. B. Keys, F. M. Andreopoulos and N. A. Peppas, Macro-
molecules, 1998, 31(23), 8149–8156.
6 S Zalipsky and J. M. Harris, in Poly(ethylene glycol): Chem-
istry and Biological Applications, ed. J. M. Harris and
S. Zalipsky, American Chemical Society, Washington DC,
1997, p. 1.
7 N. P. Desai, S. F. A. Hossainy and J. A. Hubbell, Biomater-
ials, 1992, 13(7), 417–420.
8 P. D. Drumheller and J. A. Hubbell, J. Biomed. Mater. Res.,
1995, 29, 207–215.
9 E. W. Merrill and E. W. Salzman, ASAIO J, 1982, 6, 60.
10 S. I. Jeon and J. D. Andrade, J. Colloid Interface Sci., 1991,
142(1), 159–166.
11 J. Groll, Z. Ademovic, T. Ameringer, D. Klee and
M. Moeller, Biomacromolecules, 2005, 6(2), 956–962.
12 M. Amiji and K. Park, Biomaterials, 1992, 13(10), 682–692.
Paper Biomaterials Science
858 |Biomater. Sci., 2013, 1, 850–859 This journal is © The Royal Society of Chemistry 2013
Published on 16 May 2013. Downloaded on 25/02/2016 14:31:32.
View Article Online
13 S. M. O’Connor, A. P. DeAnglis, S. H. Gehrke and
G. S. Retzinger, Biotechnol. Appl. Biochem., 2000, 31, 185–
196.
14 V. Y. Alakhov, E. Y. Moskaleva, E. V. Batrakova and
A. V. Kabanov, Bioconjugate Chem., 1996, 7(2), 209–216.
15 C.-C. Lin and K. S. Anseth, Pharm. Res., 2009, 26(3), 631–
643.
16 S. H. G. Khoo and M. Al-Rubeai, Biotechnol. Appl. Biochem.,
2007, 47,71–84.
17 S. G. Villas-Bôas, S. Mas, M. Åkesson, J. Smedsgaard and
J. Nielsen, Mass Spectrom. Rev., 2005, 24, 613–646.
18 O. Beckonert, H. C. Keun, T. M. D. Ebbels, J. Bundy,
E. Holmes, J. C. Lindon and J. K. Nicholson, Nat. Protoc.,
2007, 2(11), 2692–2703.
19 G. Schlotterbeck, A. Ross, F. Dieterle and H. Senn, Pharmaco-
genomics,2006,7,1055–1075.
20 O. Fiehn, J. Kopka, P. Dörmann, T. Altmann,
R. N. Trethewey and L. Willmitzer, Nat. Biotechnol., 2000,
18, 1157–1161.
21 L. E. McNamara, T. Sjöström, K. E. V. Burgess, J. J. W. Kim,
E. Liu, S. Gordonov, P. V. Moghe, R. M. D. Meek,
R. O. C. Oreffo, B. Su and M. Dalby, Biomaterials, 2011, 32,
7403–7410.
22 S. Kelleher, A. Jongerius, A. Loebus, C. Strehmel, Z. Zhang
and M. C. Lensen, Adv. Eng. Mater., 2012, 14(3), B56–B66.
23 M. E. Smith and E. H. Finke, Invest. Ophthalmol., 1972,
11(3), 127–132.
24 K. Dettmer, N. Nürnberger, H. Kaspar, M. A. Gruber,
M. F. Almstetter and P. J. Oefner, Anal. Bioanal. Chem.,
2011, 399, 1127–1139.
25 A. Luedemann, K. Strassburg, A. Erban and J. Kopka, Bio-
informatics, 2008, 24(5), 732–737.
26 A. Saeed, V. Sharov, J. White, J. Li, W. Liang, N. Bhagabati,
J. Braisted, M. Klapa, T. Currier, M. Thiagarajan, A. Sturn,
M. Snuffin, A. Rezantsev, D. Popov, A. Ryltsov,
E. Kostukovich, I. Borisovsky, Z. Liu, A. Vinsavich, V. Trush
and J. Quackenbush, BioTechniques, 2003, 34(2), 374–378.
27 P. B. van Wachem, T. Beugeling, J. Feijen, A. Bantjes,
J. P. Detmers and W. G. van Aken, Biomaterials, 1985, 6,
403–408.
28 Y. Tamada and Y. Ikada, J. Biomed. Mater. Res., 1994, 28,
783–789.
29 F. Zhang, E. T. Kang, K. G. Neoh, P. Wang and K. L. Tan,
Biomaterials, 2001, 22, 1541–1548.
30 M. C. Lensen, V. A. Schulte, J. Salber, M. Diez, F. Menges
and M. Möller, Pure Appl. Chem., 2008, 80(11), 2479–2487.
31 G. Tan, Y. Wang, J. Li and S. Zhang, Polym. Bull., 2008, 61,
91–98.
32 Y. Chang, W.-L. Chu, W.-Y. Chen, J. Zheng, L. Liu,
R.-C. Ruaan and A. Higuchi, J. Biomed. Mater. Res., Part A,
2010, 93(1), 400–408.
33 C. J. Kirkpatrick and C. Mittermayer, J. Mater. Sci.: Mater.
Med., 1990, 1(1), 9–13.
34 F. Dankberg and M. D. Persidsky, Cryobiology, 1976, 13,
430–432.
35 M. B. Browning and E. Cosgriff-Hernandez, Biomacromole-
cules, 2012, 13(3), 779–786.
36 V. A. Schulte, M. Diez, M. Möller and M. C. Lensen, Bio-
macromolecules, 2009, 10(10), 2795–2801.
37 K. W. Lanks, J. Biol. Chem., 1987, 262(21), 10093–10097.
38 H. Eagle, Science, 1959, 130, 432–437.
39 J. Mohberg and M. J. Johnson, J. Natl. Cancer Inst., 1963, 31
(3), 611–625.
40 J. B. Griffiths, J. Cell Sci., 1970, 6, 739–749.
41 H. Eagle, J. Biol. Chem., 1955, 214(2), 839–852.
42 L. Levintow, H. Eagle and K. A. Piez, J. Biol. Chem., 1957,
227(2), 929–941.
43 R. J. X. Zawada, P. Kwan, K. L. Olszewski, M. Llinas and
S. G. Huang, Biochem. Cell Biol., 2009, 87, 541–544.
44 G. Stabellini, G. Mariani, F. Pezzetti and C. Calastrini, Exp.
Mol. Pathol., 1997, 64, 147–155.
45 J. Luo, N. Vijayasankaran, J. Autsen, R. Santuray,
T. Hudson, A. Amanullah and F. Li, Biotechnol. Bioeng.,
2012, 109(1), 146–156.
46 K. E. Weibel, J.-R. Mor and A. Fiechter, Anal. Biochem.,
1974, 58, 208–216.
47 P. L. Yeagle, Biochim. Biophys. Acta, 1985, 822, 267–287.
48 D. A. Ford, Prog. Lipid Res., 2002, 41(1), 6–26.
49 S. P. Bruttig and W. L. Joyner, J. Cell. Physiol., 1983, 116,
173–180.
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