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Light-Responsive Hierarchically Structured Liquid Crystal Polymer Networks for Harnessing Cell Adhesion and Migration

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Extracellular microenvironment is highly dynamic where spatiotemporal regulation of cell-instructive cues such as matrix topography tightly regulates cellular behavior. Recapitulating dynamic changes in stimuli-responsive materials has become an important strategy in regenerative medicine to generate biomaterials which closely mimic the natural microenvironment. Here, light responsive liquid crystal polymer networks are used for their adaptive and programmable nature to form hybrid surfaces presenting micrometer scale topographical cues and changes in nanoscale roughness at the same time to direct cell migration. This study shows that the cell speed and migration patterns are strongly dependent on the height of the (light-responsive) micrometer scale topographies and differences in surface nanoroughness. Furthermore, switching cell migration patterns upon in situ temporal changes in surface nanoroughness, points out the ability to dynamically control cell behavior on these surfaces. Finally, the possibility is shown to form photoswitchable topographies, appealing for future studies where topographies can be rendered reversible on demand.
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Light-Responsive Hierarchically Structured Liquid Crystal
Polymer Networks for Harnessing Cell Adhesion
and Migration
Gülistan Koçer, Jeroen ter Schiphorst, Matthew Hendrikx, Hailu G. Kassa,
Philippe Leclère, Albertus P. H. J. Schenning,* and Pascal Jonkheijm*
DOI: 10.1002/adma.201606407
The extracellular matrix (ECM) continuously submits to cells
tightly regulated spatiotemporal changes in chemical, physical,
and mechanical cues. These dynamic changes in signals to cells
eventually prompt changes in cell and tissue behavior.[1] In regen-
erative medicine, various strategies have been used to engineer
materials to present ECM inspired cues to direct (stem) cell fate
and tissue function.[1b,2] Notwithstanding these achievements,
Extracellular microenvironment is highly dynamic where spatiotemporal
regulation of cell-instructive cues such as matrix topography tightly regulates
cellular behavior. Recapitulating dynamic changes in stimuli-responsive mate-
rials has become an important strategy in regenerative medicine to generate
biomaterials which closely mimic the natural microenvironment. Here, light
responsive liquid crystal polymer networks are used for their adaptive and
programmable nature to form hybrid surfaces presenting micrometer scale
topographical cues and changes in nanoscale roughness at the same time
to direct cell migration. This study shows that the cell speed and migra-
tion patterns are strongly dependent on the height of the (light-responsive)
micrometer scale topographies and differences in surface nanoroughness.
Furthermore, switching cell migration patterns upon in situ temporal changes
in surface nanoroughness, points out the ability to dynamically control cell
behavior on these surfaces. Finally, the possibility is shown to form photo-
switchable topographies, appealing for future studies where topographies can
be rendered reversible on demand.
Biointerfaces
G. Koçer, Prof. P. Jonkheijm
Bioinspired Molecular Engineering Laboratory
MIRA Institute for Biomedical Technology and Technical Medicine
and Molecular Nanofabrication Group
MESA+ Institute for Nanotechnology
Department of Science and Technology
University of Twente
7500 AE, Enschede, The Netherlands
E-mail: p.jonkheijm@utwente.nl
J. ter Schiphorst, M. Hendrikx, Dr. P. Leclère, Prof. A. P. H. J. Schenning
Functional Organic Materials and Devices
Department of Chemical Engineering and Chemistry
Eindhoven University of Technology
5612 AE, Eindhoven, The Netherlands
E-mail: a.p.h.j.schenning@tue.nl
J. ter Schiphorst, M. Hendrikx, Dr. P. Leclère, Prof. A. P. H. J. Schenning
Institute for Complex Molecular Systems
Eindhoven University of Technology
P.O. Box 513, 5600 MB, Eindhoven, The Netherlands
Dr. H. G. Kassa, Dr. P. Leclère
University of Mons (UMONS)
Laboratory for Chemistry of Novel Materials
Center for Innovation and Research in Materials and Polymers
(CIRMAP)
Research Institute for Materials Science and Engineering
Place du Parc, 20 B-7000 Mons, Belgium
The ORCID identification number(s) for the author(s) of this article
can be found under http://dx.doi.org/10.1002/adma.201606407.
the majority of biomaterials that present
these cues are static, with predefined
properties that cannot submit a changing
environment to cells at any specific point
in time. Therefore, designing new bio-
materials with the ability to change proper-
ties in time is important as such dynamic
materials would mimic more closely the
natural microenvironment of cells.[1] This
can be achieved by incorporating stimuli-
responsive elements into materials using,
for example, thermosensitivity.[1a,3] Control
over size (from the nano to micrometer
scale) and arrangement of topographical
features as one of the matrix cues is known
to have great influence on cell adhesion,
spreading, migration and differentiation,
and tissue organization.[2a,4] Therefore,
recapitulating topographical changes of a
natural matrix in situ (i.e., in presence of
cells) is highly interesting for the genera-
tion of new biomaterials. Several studies
on dynamic (i.e., in situ at a specific point in time changed,
therefore temporal changes, either irreversibly or reversibly)
topographies have used strain-responsive buckling of plasma
oxidized polymethylsiloxane thin films[5] or thermally activated
shape-memory polymer (i.e.,
ε
-polycaprolactone)[6] and strain-
responsive liquid crystal elastomers[7] in dynamic control of
(stem) cell alignment, spreading, and differentiation.
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Nevertheless, in these systems, careful selection of the stim-
ulus considering in situ experiments as well as the versatility of
surface modifications in terms of feature generation is essen-
tial. Generally, light offers flexibility and remote control, nonin-
vasiveness and physiological compatibility, i.e., when tuned in
correct doses and wavelength, to generate changes in surface
or bulk properties of biomaterials.[1a,8] In fact, light has been
extensively used to pattern and control the presentation of cell
adhesive ligands in vitro[8,9] and in vivo[10] to tune chemical
and physical properties of cell encapsulated hydrogels,[1a,3a] to
control tissue assembly and to release patterned cells from sur-
faces.[11] The conceivable benefits of employing light responsive
systems are recognized and encourage utilization to advance
dynamic surface topographies across larger areas.[12] Yet, there
are only a few examples where light has been used to form (re)
configurable topographies in polymer systems consisting of
photoswitchable molecules in order to control cell spreading
and alignment while challenges regarding in situ studies still
remain.[12,13] In a very recent example, Netti and co-workers
elegantly showed in situ spatiotemporal control of topography
to direct cell alignment on azopolymers.[14] However, so far no
study has documented if in situ light-induced changes (i.e.,
temporal changes) in surface structure effects cell migration,
which is one of the essential processes in tissue remodeling
and wound healing as well as in colonization of biomaterials
by desired cell types for successful implantation.[4f,15] Here, we
employ the adaptive and programmable nature of light respon-
sive liquid crystal polymer network (LCN) coatings[16] to achieve
new spatially arranged patterned biointerfaces for in situ tem-
poral control of surface properties to guide cell behavior. These
azobenzene based LCN systems can operate in solvent free
environments and present a versatile way to change surface
topography both in a reversible and irreversible fashion.[16,17]
We show that these LCNs are biocompatible and can be used as
bioinstructive responsive materials to control cell adhesion and
migration. Furthermore, we exploit these materials to induce
in situ temporal changes (permanent in this case) in surface
structural properties (i.e., roughness) to control cell migration.
The preparation of the responsive LCN coatings (with com-
ponents given Scheme 1) is given in Figure 1A (mesogenic
monomers, Table S1, in the Supporting
Information). A mixture of (meth)acrylate
functionalized azobenzene and liquid crys-
talline monomers were used to create a so-
called chiral nematic phase that is subse-
quently aligned in plane by shear forces and
then photopolymerized (see details in the
Supporting Information). Mask illumination
of the films lead to local trans to cis isomeri-
zation of azobenzene molecules conjugated
in the network resulting in a local formation
of protrusions (volume generation) in the
illuminated areas yielding (microscale) topo-
graphical cues for cells (Figure S1A (Sup-
porting Information) and Figure 1B–D).[17]
These surface topographies can be rendered
permanent by modifying the starting mix-
ture composition.[17a] Cell adhesion and
migration were studied either on surfaces
with predefined (fixed) microscale topographical cues or on
surfaces where at a specific point in time the surface structure
was changed in situ. By using a mask containing a hexagonal
pattern, hexagonally arranged pillars (HP) were formed. The
height of the HPs was determined for cellular studies in the
micrometer scale by conveniently varying the illumination
dose (intensity). A series of surfaces were prepared with pil-
lars ranging in height from 0.2 to 1.6 µm, having a diameter of
20 µm and spaced 20 µm from each other as was verified using
optical profilometry (Figure 1B,C, HP-0.2 µm, HP-0.3, HP-0.5,
HP-1.2, and HP-1.6 µm). Other patterns were achieved using
other masks, such as circular patterns (CP) of, e.g., 15 µm in
width and separation and with a height of 0.3 µm (CP-0.3),
demonstrating the versatility of this contactless method to fabri-
cate topographical polymeric biointerfaces (Figure 1D). As con-
trol surfaces to the films with microscale topographies, “flat”
(with respect to microscale) LCN surfaces were used that were
or were not entirely illuminated (Flat_i or Flat_ni). Upon illu-
mination the morphology of the film alternates, which is veri-
fied by a change in the absorbance properties of the polymer
films (Figure S1B, Supporting Information). Atomic force
microscopy (AFM) measurements on Flat_ni and Flat_i sur-
faces revealed an increase in surface roughness on the illumi-
nated areas changing from 9.9 ± 0.9 to 18.2 ± 0.4 nm showing
that the nanoscale topography was changed upon illumination
(Figures S1C,D and S2A,B, Supporting Information). It should
be noted that the formation of pillars further increases the
nanoroughness (vide infra). The surfaces were hydrophobic
irrespective of illumination with contact angles of 88° ± 1° and
101° ± 4° for Flat_ni and Flat_i, respectively. In order to facili-
tate protein adsorption and hence cell adhesion on these sur-
faces, all the LCN films were incubated and coated with serum
proteins overnight before performing the cell experiments.
Use of established cell lines such as NIH3T3 fibroblasts is
informative in revealing adhesion related cellular processes.[4b,14]
Adhered NIH3T3 fibroblast cells and their supported growth
on all the fabricated LCN surfaces (Figure 1E–M and Videos
S1–S9 and Figure S3 (Supporting Information)) validated the
biocompatibility of these LCN films similar to polystyrene,
which is commonly used for cell cultures. From time-lapse
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Scheme 1. Molecular structures of the materials that were used for preparing the light-respon-
sive liquid crystal polymer (Compounds are 1: Light responsive molecule, 2: Chiral dopant, 3–5:
Nematic hosts, 6: Photoinitiator, 7: Chain stopper/inhibitor).
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imaging experiments performed on the films that were incu-
bated with fibroblasts, single cell migration behavior and the
mean cell migration speed (total distance covered per time of
measurements) were determined (Figures 2 and 3). The initial
mean cell migration speed of 0.85 µm min1 observed on flat
LCN films significantly dropped to 0.32 µm min1 when such
films were illuminated entirely, while qualitatively no changes
in cell morphology were observed (Figures 2H and 3A–C and
Videos S1 and S2 (Supporting Information)). Furthermore, cell
migration patterns were derived from the time-lapse images and
plotted as a change in cell migration speed in intervals of 5 min
(Figure 3G,H). These plots not only confirm that cells became
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Figure 1. Encoding topography in films using light in LCNs to harness cell behavior. A) Schematic representation of the method to fabricate HP
topographies in LCN films by using a hexagonally patterned mask and B) 3D representation of a HP surface. C) Height profiles of the HP surfaces.
D) 3D representation of the CP surface. E–H) Phase contrast images of NIH3T3 cells after 1 d on HP-1.6, HP-1.2, HP-0.5, HP-0.2 µm, respectively.
I) SEM image of cells on a HP surface (with 15 µm diameter and separation, and 0.15 µm height) after 12 h. J) Phase contrast image of cells on CP-0.3
surface after 1 d. K,L) SEM images of cells on CP-0.3 surface after 12 h and M) phase contrast image of cells after 1 d on tissue culture polystyrene as
control surface (scale bar: 50 µm for (E–H, J, M); 10 µm for (I), and 100 µm for (K, L)).
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more static on illuminated substrates but also reveal changes in
migration speed that occurred when cells were observed over
the course of a few hours. These changes in migration speed
occurred similarly frequent, yet of a markedly lower magni-
tude upon illumination (Figure 3G,H). These observations
can be related to the increase in nanoscale surface roughness
upon illumination, as was measured using AFM (9.9 ± 0.9
versus 18.2 ± 0.4 nm, for Flat_ni versus Flat_i, respectively,
Figure S1C,D, Supporting Information). It is known that cells
are able sense nanoscale features down to 8 nm and distinguish
the size of the features by their nanopodial extensions.[4b,18] Yet,
studies also show that in general, cell attachment is influenced
by changes in mechanical properties.[19] To gain more insight in
the nanoscale topographical and mechanical properties of the
Adv. Mater. 2017, 1606407
Figure 2. Characterization of single cell migration behavior on LCN surfaces. A) AFM morphology and height image of a 0.75 µm pillar and its sur-
rounding. B) Derjaguin–Müller–Toporov (DMT) effective elastic modulus map obtained from one ImAFM scan of the 0.75 µm HP (given in Figure A)
(The color contrasts and the scale bars depict the value of the stiffness on the sample surface in GPa. The scan size is 40 × 40 µm2). Snapshots from
live cell imaging for representative cells C,D) residing on the pillars and E) stationary and in contact with the pillars on the flat surfaces on HP-0.3,
HP-1.6, HP-1.2 topographies, respectively (black arrow: a representative stationary cell). F,G) Representative cells that are motile over the pillars and
on the flat areas on HP-1.2 and HP-1.6 µm surfaces, respectively (scale bar: 50 µm). H) Single cell migration speed on different LCN surfaces. The
black circles are nonclassified cell categories, while the blue circles (open and closed) show category 1 and 2, respectively (ni: not illuminated, red
line: the mean cell speed, mean ± standard deviation, **p < 0.01 and *p < 0.05). I) Percentage of the cells on HP surfaces representing the whole cell
population counted on the surfaces in both categories.
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pillars and surrounding areas we imaged a hierarchically struc-
tured HP-0.75 µm film using intermodulation atomic force
microscopy (ImAFM) (Figure 2A,B and Figure S2 and Table S2
(Supporting Information)). Height and roughness on top of
the pillars and in their surrounding areas were determined and
compared to illuminated flat surfaces. Illumination of flat films
resulted in a 20 nm increase in nanoscale roughness (Table S2,
Supporting Information). The surfaces of illuminated pillars
were found to also be rougher (55 ± 3.3 nm) compared with
the nonilluminated surrounding areas (10 ± 1.4 nm). These
results verified that we have fabricated hybrid interfaces pre-
senting light-induced topographical patterns with significantly
increased nanoscale roughness. ImAFM inspections also reveal
simultaneously nanoscale mechanical properties of the sur-
face of the pillars and their surrounding areas (Figure 2B and
Figure S2C,D (Supporting Information)). Figure 2B is a typical
effective elastic modulus map showing an average effective
elastic modulus (0.5–0.75 GPa) of the surfaces of illuminated
Adv. Mater. 2017, 1606407
Figure 3. Characterization of surface topology and pillar height dependent switch in cell migration behavior. A) Average speed of cells on both
flat surfaces and cells on HP-0.3 µm surface in Category 1. Phase contrast images of cells after 1 d on B) flat not illuminated, C) flat illuminated,
and D,E) HP-0.3 µm surface (scale bar: 50 µm). F) Time-lapse imaging for a cell that represents Category 1 on HP-0.3 µm surface (scale bar: 50 µm).
G–J) Cell migration patterns as change in single cell speed at every 5 min for four representative cells on different LCN surfaces.
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pillars, which was found lower than on nonilluminated
surrounding areas (1.2 GPa) (see the Supporting Information
for details). Even though the elastic modulus has decreased
upon illumination, we believe that both illuminated and nonil-
luminated areas present stiff matrices to cells to a similar
extent due to their high elastic modulus (in GPa) compared to
elastic natural matrix (elastic modulus ranging from a few hun-
dred Pa to a few hundred kPa).[19,20] Forces generated by cells
are in the range of 1–5 nN µm2 and cells would need to pull
against and deform the underlying substrate to be able to sense
and respond to stiffness.[19a,20,21] Therefore, our hierarchical
interfaces allow us to relate the effect of nanoscale roughness
and micrometer scale topographical cues independently of the
stiffness of the surfaces.
The derived mean cell migration speeds on LCN films with the
HP topographical cues revealed that surfaces with 0.3 µm high
pillars (HP-0.3 µm) induced a significantly lower mean cell speed
of 0.23 µm min1 when compared to the HP-0.5, HP-1.2, and
HP-1.6 surfaces, which had a mean cell speed of 0.87, 0.81,
and 0.87 µm min1, respectively (Figure 2H and Videos S4–S7
(Supporting Information)). Interestingly, the latter topogra-
phies induced cell speeds in the same range as observed on the
nonilluminated flat LCN films, while the cell speed observed on
the HP-0.3 topography is similar to that on the illuminated flat
LCN films. The mean cell speed of 0.52 µm min1 observed on
HP-0.2 topographies represents an intermediate case (vide infra).
As a control surface for cell adhesion and migration, a speed of
0.55 µm min1 was observed on classical tissue culture polystyrene
surfaces, which lies in between the values observed for both flat
LCN surfaces (Figure S4 and Video S9, Supporting Information).
To closer inspect how the HP topographies were harnessing
the spatiotemporal adhesion and organization of cells on these
surfaces during migration, the live cell imaging data were ana-
lyzed in more detail (see the Experimental Section). From the
analysis, two modes of cell behavior were identified; cells in cat-
egory 1 were mainly stationary with mean cell speeds as low
as 0.15 µm min1 and these cells were mainly in contact with
the pillars while having minimal contact with the areas sur-
rounding the pillars (Figure 2C,E); cells in category 2 were gen-
erally mobile with mean cell speeds as high as 1.13 µm min1
and these cells were having minimal contact with pillars and
were mainly moving across areas surrounding the pillars
(Figure 2F,G). Much to our surprise, the analysis shows that the
majority (84%) of the cells on HP-0.3 µm belong to static, cat-
egory 1 type cells (Figure 2H,I), while the majority (70%–87%)
of cells on films with higher pillars (HP-0.5, 1.2, and 1.6 µm)
fall to mobile, category 2 type cells. These category 2 cells
migrate with no significant differences on the HP-0.5, 1.2, and
1.6 topographies (Figure 2H), yet they do migrate significantly
faster when compared to the category 1 cells on HP-0.3 topog-
raphies. It is known that higher cell speed can be related to the
persistence in directional movement.[4f,22] However, trajectory
analysis of cells in category 2 on all HP surfaces did not reveal
any direct correlation of cell speed with persistence and direc-
tionality (Figure S5, Supporting Information). As the average
speeds of the two categories correspond with the speed of the
illuminated and nonilluminated flat surfaces, we tentatively
assign the remarkable, static versus mobile appearance of cells
to the extent that cells were in contact with a rougher surface
existing on illuminated parts of the films versus their contact
with a smoother surface existing on nonilluminated parts of the
films. The cell migration behavior that was imaged on HP-0.2
topographies could not be categorized due to a lack of resolving
the topography during imaging (black circles in Figure 2H).
Yet, cells on HP-0.2 µm were spread over both pillars and
nonilluminated areas indicative of cells that fall into category 2
(Figure 1H and Video S3 (Supporting Information)).
Most notable are the consequences for cell migration
behavior when switching from flat surfaces to a HP-0.3 topog-
raphy. Category 1 cells on HP-0.3 topographies were adherent
and residing only on the pillars, mainly elongating over the pil-
lars for extended periods of up to 5 h (Figure 3D–F) in agree-
ment with the observed contact of cellular protrusions on the
rougher pillars (Figure 1I). In contrast, cells on the flat LCN
surfaces, irrespective of illumination, were spread, but not as
elongated, which is typical for cell morphologies on 2D sur-
faces (Figures 3B,C and 1M). This clearly points toward topog-
raphy guided cell morphology, which is known to influence cell
motility.[2a,4d–f] Intriguingly, even though cells were residing
on illuminated parts of the topographic films, presumably due
to increased roughness, the mean cell migration speed of the
category 1 type cells on HP-0.3 significantly dropped further
to 0.15 µm min1 when comparing to the cell migration speed
of 0.32 µm min1 on flat, illuminated films (Figure 3A). The
migration pattern plots for HP-0.3 topographies reveal that
this category of cells became even more static with lower and
less frequent changes observed in cell speed (Figure 3H,I). In
strong contrast, cells on HP-1.2 topographies yielded a dramatic
shift in the cell migration pattern to a highly dynamic behavior
with increasing frequencies and magnitudes of changes in cell
migration speed over time (Figure 3I,J and Figure S6 (Sup-
porting Information)).
Next to the HP topographic patterned films, single cell migra-
tion speed and cell behavior were also imaged and analyzed on
the circular patterns of comparable heights and spacing as the
HP patterns (Video S8, Supporting Information). This anal-
ysis similarly revealed the existence of two distinct cell popu-
lations with cells aligning on the patterns of 0.3 µm in height
and 15 µm spacing. These results show that cell adhesion and
migration was guided on a different surface geometry indicating
that the observations are generic (Figure 1J–L and Figure S7
(Supporting Information)). These results demonstrate also the
versatility of employing light responsive LCN films as new
biointerfaces to direct cell movement and migration speed.
The changes in cell migration patterns are clearly associated
with synergistic cell contact guidance by the micrometer scale
topographies and the nanoscale roughness both of which are
consequences of the light-induced isomerization of the azoben-
zene moieties in the bulk of the film material.[2a,4b] Switching
cell migration behavior by switching the surface structure have
important implications regarding in situ dynamic experiments
where cell migration could be differentially harnessed to guide
cells to specific locations on the surface.
To explore the possibility to guide cell migration and col-
lect these migrating cells in local spots, the following surface
topographic pattern was designed. Films were prepared with
flat, nonilluminated areas surrounded by HP-1.1 topographies
(Figure 4A,B and Figure S8A (Supporting Information)). Our
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observations on such mixed surface-structured films revealed
that the cell migration speed was similar on both areas of the
surface and in agreement with the speeds and trends observed
on either of the individual, nonmixed surface-structured sur-
faces (Figure 2H and Figure S8B and Video S10 (Supporting
Information)). However, much to our surprise, the majority of
the cells (82%, 9 out of 11 cells that were monitored for 4 h)
starting from the pillar area, entered the flat area and remained
in this area with a spread morphology (Figure 4B). This result
shows the possibility to spatially control the localization of the
cell population through cell migration on these surfaces, which
has important implications in colonization of cells on biomate-
rials to enhance their performance.[15]
With these results in hand, consequences of in situ temporal
(i.e., changes occur in time in the presence of cells) light-induced
permanent surface structural changes on the cell behavior
were studied representing a first step in exploring the poten-
tial of these materials in dynamically controlling the surface
topography. For this purpose, cells were seeded on flat, nonil-
luminated surfaces and before illumination of the substrate the
typical response of cells on these surfaces was
observed (Figures 3G and 4D). For this type
of experiments, a critical consideration is the
biocompatibility of the illumination dose.[1a]
By illuminating the film from the bottom,
light penetration measurements using an
intensity detector (details are given in the
Supporting Information) showed no UV pen-
etration through the samples to the cells indi-
cating that UV light was absorbed by the film.
According to this measurement, the films
were exposed to light of
λ
= 390–440 nm for
10 min (35 mW cm2), which was sufficient
to induce the structural changes in rough-
ness. According to AFM measurements on
this sample an increase in surface roughness
from 9.0 ± 1.2 to 11.1 ± 2 nm was observed
upon illumination for 10 min (Figure S9,
Supporting Information), where a change in
cell migration can be expected.[4b,f,18,23]
Upon illumination, cells were monitored
for 12 h before measuring the cell migra-
tion speed, to observe and confirm their
viability after illumination. Live cell imaging
data revealed that 87% of the cells remained
viable within this time (Videos S11 and S12,
Supporting Information). Changes in cell
migration speed occurred less frequent and
to a smaller extent with a decrease in mean
cell speed from 1.21 to 0.54 µm min1 after
illumination showing a shift toward a more
static behavior (Figures 3H and 4E and
Figure S10 (Supporting Information)). These
observations are in accordance with the ex
situ experiments (Figure 3A) where speed
decreased with increased roughness.
In conclusion, consequences for cell
migration on light-induced topographi-
cally patterned polymer network films were
studied as function of micrometer scale topographical height
and pattern and nanoscale surface roughness on similarly stiff
surfaces for cells. Cell motility patterns could be switched from
static to highly mobile by increasing the height of the pillars or
from dynamic to moderately static by increasing the nanoscale
roughness or to highly static on a selective topographic pattern.
In situ temporal experiments further demonstrated the effect of
nanoscale changes. Preliminary experiments on patterned LCN
surfaces showed that it is also possible to form photoswitchable
(reversible) topographies on demand (Figures S11 and S12, Sup-
porting Information) at the nanoscale which is another impor-
tant step for in situ control of cell behavior. These results demon-
strate a new approach in engineering bioinstructive light respon-
sive surface structures. Future experiments aim at in situ revers-
ible studies as well as manipulation of microscale topographies
where light responsive elements that are switchable at longer
wavelengths can be used to match the volume generation prop-
erties with cytocompatible illumination doses.[24] The freedom
that is offered in this system will enable us to generate mate-
rials that are more closely recapitulating the dynamic natural
Figure 4. Spatial and temporal switches in cell behavior on the same LCN surface. A) 3D
representation of the mixed surface with a Flat_ni region surrounded by HP-1.1 topographies.
B) Phase contrast image of NIH3T3 cells on LCN surfaces presenting a flat not illuminated area
surrounded by 1.1 µm high HPs after 3 d of culture. C) Live cell imaging for a representative cell
going from the pillar area to the flat area (scale bar: 50 µm). D,E) In situ switch in cell migration
patterns on flat LCN surfaces as change in single cell speed at every 5 min.
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microenvironment not just giving better insights in funda-
mental biological processes but will also enable us to enhance
biomaterial performance for regenerative medicine applications.
Experimental Section
Preparation and Characterization of Liquid Crystal Polymer Network
Surfaces: Detailed information on preparation of light responsive
LCN materials is given in the Supporting Information. Material
characterization was performed with UV–vis spectroscopy, AFM, optical
profilometry, and contact angle measurements. Details of surface
characterization are also given in the Supporting Information.
Characterization of Cell Behavior: NIH 3T3 (ATTC CRL-1658) cells
were seeded at a density of 5000 cells cm2 on the surfaces and time
lapse images were taken every 5 min using Cytomate live cell imaging
equipment (Eindhoven, The Netherlands). Cell trajectories and cell
migration speed were derived using the MTrackJ plugin of ImageJ
(NIH) by following individual cells. In situ switching experiments were
performed at room temperature for 10 min, after 1 d of cell adhesion,
in live cell imaging medium. Live cell imaging was performed as
explained above to observe cell migration before and after illumination
of the substrates. Cell adhesion and organization on the surfaces were
analyzed with phase contrast and scanning electron microscopy (SEM).
See the Supporting Information for details.
Statistics: Data are presented as mean ± standard deviation and cell
migration data were analyzed using Kruskal–Wallis one way-ANOVA
for statistical significance. Multiple comparison tests were performed
with Mann–Whitney test by adjusting
α
level downward according to
Bonferroni correction comparing all groups of interest.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
P.J. and G.K. thank NWO (VIDI Grant No. 723.012.106) for funding.
J.t.S. and A.P.H.J.S. thank EU FP7 for funding (NAPES Project
No. 604241). M.H. thanks NWO (TOP PUNT Grant No. 10018944) for
funding. P.L. thanks FEDER (BELSPO-PAI VII/5), P.L. and H.G.K. thank
FRS-FNRS PDR (ECOSTOFLEX). Danqing Liu, Sisi Tang, Erik F.G.A.
Homburg, Marc P.F.H.L. van Maris, Dick J. Broer, Jenny Brinkmann,
Koen Nickmans, and Jeffrey N. Murphy are acknowledged for helpful
discussions and assistance during measurements.
Conflict of Interest
The authors declare no conflict of interest.
Keywords
biointerfaces, cell–biomaterial interactions, liquid crystals, stimuli-
responsive topographies
Received: November 26, 2016
Revised: March 20, 2017
Published online:
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