1 Department of Chemical Engineering, School of Veterinary Medicine, University
of Wisconsin, Madison, WI 53706, USA
2 Department of Surgical Sciences, School of Veterinary Medicine, University of
Wisconsin, Madison, WI 53706, USA
3 Department of Biomolecular Chemistry, School of Medicine, University of
Wisconsin, Madison, WI 53706, USA
Authors for correspondence (e-mail:
nealey{at}engr.wisc.edu,
murphyc{at}svm.vetmed.wisc.edu)
Accepted 21 January 2003
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Summary |
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This work documents that biologic length-scale topographic features that model features encountered in the native basement membrane can profoundly affect epithelial cell behavior.
Key words: Cell-substrate interactions, Substrate topography, Grooves and ridges, Contact guidance, Focal adhesions, Nanobiology
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Introduction |
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Anisotropic topographic features have been shown to induce many cell types
to align and migrate along the direction of the anisotropy, a phenomenon
called contact guidance (Flemming et al.,
1999). We investigated whether human corneal epithelial cells
sense and react to nanoscale substrate topographies by evaluating the cellular
responses to anisotropic topographic features with dimensions in the nanometer
range. Cell elongation and alignment on grooves and ridges of nanoscale
dimensions was compared with the morphology and orientation of cells cultured
on smooth substrates and on substrates with micrometer-sized grooves and
ridges. The smallest topographic features tested (70 nm) were more than 100
times smaller than the width of a single cell. The substrates had uniform
surface chemistry, ensuring that chemical anisotropies did not contribute to
the observed cellular responses.
Various cell types display contact guidance when cultured on groove and
ridge patterns with lateral dimensions in the micrometer range
(Clark et al., 1990;
den Braber et al., 1995
;
den Braber et al., 1996a
;
den Braber et al., 1996b
;
den Braber et al., 1998
;
Flemming et al., 1999
;
Matsuzaka et al., 2000
;
van Kooten et al., 1998
;
Walboomers et al., 1999a
;
Walboomers et al., 1999b
;
Wojciak-Stothard et al.,
1995b
; Wojciak-Stothard et
al., 1996
). Profound differences in cytoskeletal organization have
been found between cells elongated and aligned along these topographic
features and cells cultured on smooth surfaces. Focal adhesions are adhesive
structures containing aggregates of transmembrane proteins called integrins
that link the actin cytoskeleton to extracellular matrix proteins. Focal
adhesions and actin microfilament bundles
(Britland et al., 1996
;
den Braber et al., 1998
;
Matsuzaka et al., 2000
;
Meyle et al., 1994
;
Walboomers et al., 1998
) and
microtubules (Oakley and Brunette,
1995a
; Oakley et al.,
1997
) have all been found to align along micrometer-sized grooves
and ridges, in cells aligned along these topographic features. On smooth
substrates, cells and cytoskeletal elements did not display any preferred
orientations.
The sequence of cytoskeletal events and the relative importance of the
actin and microtubule systems in the mechanism of contact guidance remain
unclear. Oakley and Brunette (Oakley and
Brunette, 1993) proposed that the first event in the reaction of
cells to grooves and ridges is the alignment of microtubules, 20 minutes after
cell plating. However, the detection of actin condensations along the
groove/ridge boundaries 5 minutes after cell adhesion suggested that actin
aggregation rather than microtubule alignment is the first and primary event
in the contact guidance mechanism
(Wojciak-Stothard et al.,
1995a
). Nonetheless, contact guidance was observed in cells
cultured on micrometer-wide grooves and ridges where either the microtubule or
the microfilament (Oakley and Brunette,
1995b
; Oakley et al.,
1997
; Walboomers et al.,
2000
; Wojciak-Stothard et al.,
1995a
) systems were disrupted by cytoskeletal poisons.
Interestingly, when submicrometer features were investigated (500 nm wide
grooves and ridges), functional microtubules were found to be necessary to
induce contact guidance (Oakley et al.,
1997
).
Focal adhesion formation and alignment has been proposed to play an
important role in the mechanism of contact guidance. Cell and cytoskeletal
alignment has generally been found to be more pronounced on patterns with
ridge widths between 1 and 5 µm than on grooves and ridges with larger
lateral dimensions (den Braber et al.,
1995; den Braber et al.,
1996a
; den Braber et al.,
1996b
; den Braber et al.,
1998
; Matsuzaka et al.,
2000
; Meyle et al.,
1994
). Moreover, on the narrower features, focal adhesions were
almost exclusively located on the tops of the ridges and were aligned along
these substrate features. O'Hara and Buck
(Ohara and Buck, 1979
)
proposed that on closely spaced grooves and ridges focal adhesion formation is
constrained to the top of the ridges because cell membrane stiffness leads to
the bridging of the grooves. Oblong focal adhesions 1-10 µm long
(Riveline et al., 2001
) orient
along the direction of the ridges to maximize their contact area, leading to
an alignment of microfilament bundles and of the cell body as a whole
(den Braber et al., 1996b
;
den Braber et al., 1998
;
Meyle et al., 1994
). This
explanation has been questioned on the basis of observations that focal
adhesions can bend around groove/ridge boundaries
(Walboomers et al., 1998
).
Nonuniformities in protein deposition on surfaces with anisotropic
topographies have also been proposed to play a role in contact guidance
(den Braber et al., 1998;
von Recum and van Kooten,
1995
). According to this hypothesis, the topographic
discontinuities have different surface energy and act as sites of preferred
protein deposition, creating patterns of proteins along the topographic
patterns. Walboomers and colleagues have not found a preference for focal
adhesion formation along the groove/ridge boundaries
(Walboomers et al., 1998
),
suggesting that adhesive proteins do not colocalize preferentially with the
surface discontinuities. Furthermore, cells have been shown to exhibit contact
guidance when cultured on anisotropic substrates without sharp discontinuities
(Walboomers et al., 1999a
).
Although there is no conclusive evidence that the topographic features affect
passive deposition of proteins, the assembly of fibronectin (Fn) and
vitronectin (Vn) filaments by fibroblasts has been reported to occur along the
substrate features (den Braber et al.,
1998
). These filaments coaligned with actin microfilaments and
were able to span grooves and ridges. Substrate discontinuities have also been
proposed to cause cell alignment through mechanical interactions with the cell
membrane that induce actin and vinculin condensations along the groove/ridge
edge (Wojciak-Stothard et al.,
1996
; Wojciak-Stothard et al.,
1995a
; Wojciak-Stothard et
al., 1995b
).
There are a few reports of contact guidance on submicrometer features; most
notably, Clark and colleagues (Clark et
al., 1991) found that fibroblast and epithelial cell lines aligned
along grooves and ridges 130 nm wide. Similar substrates were found to induce
alignment of oligodendrocytes but not of rat hippocampal or cerebellar neurons
(Webb et al., 1995
).
In the present study we have found that ridges 70 nm wide induced human corneal epithelial cells to elongate and align along the topographic features. This is the smallest feature size that has been reported to induce contact guidance. The percentage of aligned cells was similar for feature pitches ranging from 400 nm (70 nm wide ridges) to 2 µm, but increased with groove depth. Cells on smooth substrates were mostly round, as were those cells on the patterned substrates that were not elongated and aligned along the grooves and ridges. Additionally, the presence of serum in the culture medium increased the percentage of aligned cells, indicating that topography can act synergistically with other cellular inputs.
The range of ridge widths included in the present study (from 70 nm to 1900
nm) encompasses the reported lateral dimensions of the focal adhesions
(between 250 nm and 500 nm) (Ohara and
Buck, 1979). Therefore, the morphology of focal adhesions on these
groove and ridge patterns can be used as an indicator of the threshold in
topographic lateral dimensions where cell responses to nanotopographies differ
from those to topographic features of larger dimensions. Mature focal
adhesions and stress fibers were observed only sporadically. The observation
of cells elongated and aligned along the topographic features without focal
adhesions and associated stress fibers suggests that assembly of these
structures is not necessary in the mechanism of contact guidance of epithelial
cells. When focal adhesions were observed, the width of the focal adhesions
was controlled by the width of the ridges on the underlying substrate. Focal
adhesions in cells cultured on substrates with ridge widths of biomimetic
dimensions (70 nm) had significantly smaller widths than focal adhesions in
cells cultured on substrates with wider ridges or on smooth substrates. This
altered focal adhesion architecture on the nanostructured substrates may have
implications in focal adhesion properties such as the tension applied on the
substrate or the intracellular signaling cascades generated.
We have established that human corneal epithelial cells are responsive to topographical cues of nanoscale dimensions. Nanoscale substrate topography is therefore a cellular input and may be instrumental in obtaining desired cell behaviors, when presented in context with other inputs. Our findings may have an impact on the design of systems for cell culture, tissue engineering and the development of implantable prosthetics.
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Materials and Methods |
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These resist patterns were transferred to the underlying silicon in a helicon etching tool (Center for Plasma Aided Manufacturing, University of Wisconsin-Madison). The resist acted as a mask, allowing only the exposed silicon areas to be etched. The gases used were SF6 and C2H2F4, both with flow rates of 18 sccm. The pressure was 2 mTorr and the antenna power was 1.5 kW. The power applied to the wafer chuck was 15 W, resulting in a bias voltage of 35 V. The antenna discharge, as well as the wafer chuck power, was pulsed at a frequency of 33.3 kHz with 50% duty cycle.
The resist remaining after etching was removed by immersing the wafers in piranha solution (7/3 (v/v) of 98% H2SO4/30% H2O2) at 110°C for 30 minutes. The wafers were then copiously rinsed with deionized water. The native silicon oxide layer on the wafers was removed by dipping the wafers in buffered oxide etch (BOE, Arch Chemicals, Norwalk, CT). Following this treatment, the wafers were coated with a layer of silicon oxide in a low pressure chemical vapor deposition reactor (LPCVD, University of Wisconsin-Madison). Oxygen and tetraethylorthosilicate were the gases used; the pressure was 1 Torr and the temperature was 675°C. When samples were re-used, they were first cleaned with piranha solution as described above. The silicon oxide coating was removed by treating the substrates with BOE. The substrates were then re-coated with silicon oxide in the LPCVD reactor.
The patterned wafers were cut with a diamond saw (MicroAutomation 1006, Woodcliff Lake, NJ) into chips containing patterned fields separated by smooth areas. These chips were glued to the bottom of 24-well plates (Becton Dickson, Franklin Lakes, NJ) using silicone aquarium sealant (Perfecto Manufacturing, Noblesville, IN). They were allowed to dry in a laminar flow hood for at least 24 hours and were immersed in deionized water for an additional 24 hours. The samples were then rinsed three times for 10 minutes with deionized water and sterilized by immersing in ethanol for 30 minutes and air-drying in a hood. Finally, they were rinsed three times with sterile PBS for 10 minutes.
Cell culture
Human corneal epithelial cells were harvested from corneas donated by the
Lions Eye Bank of Wisconsin (Madison, WI) or the Missouri Lions Eye Bank
(Columbia, MO). The corneal buttons were trimmed to exclude any scleral or
limbal regions. The buttons were immersed in a dispase solution (1.2 units/ml,
Boehringer Mannheim, Germany) and placed in an incubator for 4 hours. The
epithelial cells became loosely adherent and could be removed by gently
rubbing the corneas with a pipette tip. The resulting cell suspension was
centrifuged and the cells were re-suspended in EpilifeTM basal
medium (Cascade Biologics, Portland, OR) with a growth supplement of defined
composition. The growth supplement contained purified bovine serum albumin,
purified bovine transferrin, hydrocortisone, recombinant human insulin-like
growth factor type-1, prostaglandin and recombinant human epidermal growth
factor (EDGS, Cascade Biologics). This cell suspension was transferred to T25
cell culture flasks (Becton Dickson), previously coated with a mixture of
fibronectin, collagen and albumin in a buffer (FNC coating mix, Biological
Research Faculty and Facility, Inc., Ijamsville, MD). Each flask contained
cells from two to four corneas. The cells were incubated at 37°C and 5%
CO2 and were harvested using 0.025% trypsin/0.01% EDTA (Cascade
Biologics), after reaching approximately 80% confluency. Neutralization was
done with a phosphate-buffered saline solution containing 0.025% purified
soybean trypsin inhibitor (Cascade Biologics). Cells were centrifuged and
resuspended in a 1:1 mixture of Dulbecco's modified Eagle medium and nutrient
mixture F-12 (D-MEM/F-12, Invitrogen, Carlsband, CA) with 0.5% dimethyl
sulfoxide (Sigma Chemical Co.). This basal medium was supplemented or not with
10% (v/v) of fetal bovine serum (FBS, Sigma Chemical Co.). Next, cells were
plated at a density of 8500 cells/cm2 in 24-well plates containing
the patterned silicon chips. They were incubated at 37°C and 5%
CO2, for 12 hours.
The medium was removed at the end of the incubation time and the cells rinsed with Dulbecco's phosphate-buffered saline (DPBS) (BioWhitaker, Walkersville, MD). The cells were fixed in 4% paraformaldeyde (Electron Microscopy Sciences, Washington, PA), 5% sucrose in 10 mM phosphate buffer at a pH of 7.4 and at room temperature, for 20 minutes. The cells were then permeabilized with 0.1% Triton X-100 in DPBS for 5 minutes and immersed in 1% bovine serum albumin in DPBS for 20 minutes. Next, the cells were incubated with 5 µg/ml of TRITC-phalloidin (Sigma Chemical Co.) in DPBS for 30 minutes. TRITC-phalloidin stains filamentous actin, and we found that we could obtain an accurate representation of the cell outline with this stain, as confirmed by using simultaneously a cell membrane stain such as thiosemicarbazide (Molecular Probes, Eugene, OR). Next, the cells were immersed three times in DPBS for 10 minutes. Cell nuclei were then stained using 90 nM of DAPI (Molecular Probes) in DPBS for 10 minutes followed by rinsing again three times in DPBS. Finally, the substrates were glued onto glass slides and Prolong (Molecular Probes) mounting medium was added to the cell side of the substrates before covering them with glass coverslips.
Vinculin staining was used as a marker for focal adhesions. After staining F-actin as described above, cells were incubated with mouse anti-human vinculin antibody (Sigma Chemical Co.) for 45 minutes at 37°C. The secondary antibody used was donkey anti-mouse immunoglobulin G (Jackson ImmunoResearch Laboratories, West Grove, PA).
Quantification of cell shape and orientation
The morphology of human corneal epithelial cells cultured on patterned
substrates was assessed by analyzing the shapes of all cells adhering to a
1.03x1.30 mm2 region in the center of each of the patterned
fields. A similar region was also analyzed on a smooth area of the same
sample. Images of the stained cells were obtained from an epifluorescence
microscope (Nikon TE300, Melville, NY) with an objective magnification of
20x, and montages of 2x2 images were created. Additionally, images
were also obtained of cells that had adhered to the bottom of the 24-well
plates (Tissue Culture Polystyrene, TCPS). All images were converted to binary
images using MetamorphTM software (Universal Imaging Corporation,
Downingtown, PA). The area and perimeter of all cells in the images were then
automatically measured. The length of the cells, defined as their longest
cord, and the cell breadth, the longest chord perpendicular to the length,
were also obtained from MethamorphTM. Cell elongation was defined
as the ratio between the length and breadth of each cell. The angles between
the length of the cells and the patterns were also obtained. The cells were
considered aligned with the grooves when this angle was less than 10°. The
DAPI images assisted in determining whether cells formed clumps or were
isolated. All cells in contact with other cells were manually removed from the
data sets. All experiments were repeated and ten samples were used in each
trial. This resulted in the analysis of over 600 cells for each of the pattern
dimensions and culture conditions.
Images of cells stained for vinculin were acquired at a magnification of 100x. Between 40 and 50 focal adhesions of cells cultured on each of the substrate topographies and on the smooth surfaces were manually traced and analyzed using NIH Image J software.
Time-lapse microscopy
Cells were seeded on the silicon substrates and incubated for 2 hours at
37°C and 5% CO2. The substrates with adherent cells were then
placed on glass coverslip shims set in a glass-bottom dish filled with medium,
with the cell side facing down. Images were acquired in an inverted microscope
(Zeiss Axiovert 200M) every 30 minutes until 12 hours had elapsed since the
cells had been seeded. Four 0.65x0.55 mm2 fields
(magnification 20x) were sequentially imaged on patterns of grooves and
ridges on a 400 nm pitch and on smooth substrates. All the cells not in
contact with other cells (28 cells on 400 nm pitch patterns and 19 cells on
smooth silicon substrates) were then manually traced and measured using Image
J software.
Imaging of cell morphology using scanning electron microscopy
Human corneal epithelial cells were rinsed in 0.1 M cacodylate buffer for
10 minutes and fixed in 3% glutaraldehyde in cacodylate buffer (Tousimis,
Rockville, MD) for 2.5 hours. The cells were then rinsed in cacodylate buffer
and post-fixed in 1% osmium tetroxide for 1 hour (Tousimis). Next, the cells
incubated in 1% tannic acid and rinsed in cacodylate buffer. Finally, cells
were dehydrated in graded ethanols, immersed in hexamethyldsilazane for 10
minutes, coated with 2 nm of platinum and imaged in a Leo 1530 field emission
scanning electron microsope (Leo Electron Microscopy, Thornwood, NY)
(Dalton et al., 2001a). Cells
were qualitatively examined for overall cell shape, orientation relative to
the underlying grooves and ridges and for the presence and morphology of
filopodia and lamellipodia.
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Results |
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Human corneal epithelial cells elongate and align along 70 nm wide
ridges
A subpopulation of the human corneal epithelial cells cultured on
substrates patterned with 70 nm wide ridges and 600 nm deep grooves, on a 400
nm pitch, were elongated and aligned along the direction of the patterns, 12
hours after cell seeding (Fig.
1a). By contrast, cells cultured on the smooth substrates were
mostly round (Fig. 1b). The
angle between the longest chord in a cell and the direction of the patterns
was designated as the cell orientation angle. Cells were considered to be
aligned with the substrate patterns when the cell orientation angle was less
than 10° (Clark et al.,
1990). Approximately 35% of the cells cultured on substrates with
70 nm wide ridges and 600 nm deep grooves, on a 400 nm pitch, were aligned
with the substrate topographies. On smooth substrates (SiOx and TCPS), the
distribution of cell orientation angles was uniform, indicating that cells
were randomly oriented (Fig.
2).
|
|
Cells were either elongated and aligned with the grooves and ridges or were approximately round. We defined cell elongation as the ratio between the longest chord in the cell (length) and the longest chord perpendicular to it (breadth). There was no correlation between cell elongation and cell orientation angle for cells cultured on smooth SiOx and TCPS as cells had an almost round morphology (average elongation approximately 1.3). The average elongation of nonaligned cells on patterned substrates (cells with orientation angles between 10° and 90°) was equivalent to the average elongation of cells cultured on smooth substrates. By contrast, aligned cells were on average significantly more elongated than nonaligned cells cultured on the patterned substrates or cells cultured on smooth substrates (Fig. 3). Therefore, cell elongation correlated strongly with cell alignment on the patterned substrates, where more than 80% of the cells that were considerably elongated (elongation higher than 2) were also aligned along the substrate patterns (Fig. 4).
|
|
Cells extend and retract lamellipodia along the direction of the
patterns
On substrates patterned with grooves and ridges on a 400 nm pitch, cells
periodically extended and retracted lamellipodia along the direction of the
patterns, as observed with time-lapse microscopy
(Fig. 5). Although lamellipodia
protruded preferentially along the pattern direction, protrusions
perpendicular to the patterns were not prohibited. Cells sometimes acquired a
round morphology after retraction of lamellipodia. Furthermore, there was a
subpopulation of cells that did not spread and remained round. On smooth
substrates, lamellipodial protrusions had random orientations and cells were
mostly round.
|
The centroids of cells cultured on the patterned substrates remained largely stationary for 12 hours after cell seeding (Fig. 6a). Cell trajectories during this time were longer on the smooth than on the patterned substrates (Fig. 6b). The mean square displacement on the smooth substrates was approximately four times larger than on the patterned substrates.
|
Cells appeared rounded up 2 hours after seeding, on the patterned substrates. On the smooth substrates, many cells had started to spread by this time and the average cell area was larger than on the patterned substrates. Average cell areas remained larger on the smooth than on the patterned substrates, for the duration of the experiment.
Pattern-induced alignment of corneal epithelial cells is more
affected by groove depth than pattern pitch
On substrates with 600 nm deep grooves, the percentage of aligned cells
after 12 hours of incubation was constant on patterns with pitches ranging
from 400 nm to 2000 nm. A drop off in cell alignment was observed for the
largest pattern pitches (4000 nm). On 150 nm deep grooves, cell alignment was
independent of pattern pitch. The percentage of aligned cells was
significantly lower on 150 nm than on 600 nm deep grooves, for pitches up to
2000 nm (Fig. 7). On the smooth
surfaces (SiOx and TCPS), the distributions of cell orientation angles were
consistent with randomly oriented populations of cells, as described above. On
substrates with both 150 nm and 600 nm deep grooves, the average elongation of
nonaligned cells was similar to the average elongation of cells on the smooth
substrates. On 600 nm deep grooves, for all pitches tested, cells that were
aligned along the topographic features had higher elongations on average than
cells on smooth surfaces. However, on 150 nm deep grooves the average
elongations of aligned cells and of cells on smooth surfaces were not
statistically different. This is consistent with the reduced number of
elongated cells found on the shallower surfaces.
|
The average area of elongated cells (elongation higher than 1.3) was higher than the area of round cells (elongation lower than 1.3) on both 600 nm and 150 nm deep grooves, for all pattern pitches. The average areas of both elongated and round cells on any of the patterned surfaces were lower than on the smooth surfaces. Moreover, cell areas on TCPS were higher than on smooth SiOx (Fig. 8).
|
Effects of serum in the culture medium
The absence of serum in the culture medium resulted in lower levels of
corneal epithelial cell alignment compared with when the medium was
supplemented with 10% FBS, on surfaces with equivalent pattern dimensions, on
600 nm deep grooves (Fig. 9).
The pitch of the patterns did not affect the percentage of aligned cells.
Aligned cells were more elongated on average than cells on smooth surfaces, on
all pattern pitches. Elongated cells had larger areas than round cells on all
surfaces. Interestingly, cells grown in the absence of serum had the same
surface area on smooth silicon as on TCPS, contrary to what was observed when
serum was present in the culture medium
(Fig. 10).
|
|
Distribution of filopodia and lamellipodia is affected by substrate
topography
Scanning electron microscopy (SEM) revealed that cells were covered with
numerous microvilli and formed both lamellipodia and filopodia. On all the
patterns tested, filopodia were able to adhere to both grooves and ridges. The
topographic features frequently guided the filopodial orientation
(Fig. 11). Lamellipodia
descended into the grooves at the cell edge along the topographic features, on
both narrow (330 nm) and wide (2100 nm) grooves. At the cell edges
perpendicular to the patterns, lamellipodia were able to adhere to the floor
of the grooves that were 2100 nm wide but not 330 nm wide, on both 150 nm and
600 nm deep grooves (Fig. 12).
On 2100 nm wide and 150 nm deep grooves, lamellipodia were frequently observed
to conform to the topographic features.
|
|
The width of focal adhesions is controlled by the ridge width
Mature focal adhesions and stress fibers were observed only sporadically in
cells cultured on both patterned and smooth substrates. Focal adhesions were
observed in cells cultured in DMEM/F12 medium with or without serum. Cells
cultured on smooth silicon formed focal adhesions and stress fibers with no
preferred orientations. On the patterned substrates focal adhesions and stress
fibers aligned along the topographic features
(Fig. 13). The measured width
of the focal adhesions was dictated by the width of the ridges on the
underlying substrate. Focal adhesions formed in cells cultured on substrates
with 70 nm wide ridges were significantly narrower than focal adhesions in
cells cultured on substrates with ridge widths of more than 400 nm. On
substrates with 250 nm wide ridges the measured width of focal adhesions was
intermediate between the widths found on 70 nm and 400 nm wide ridges. When
the ridge width increased from 650 nm to 1900 nm, the measured width of the
focal adhesions remained constant. However, on the wider ridges the
variability in focal adhesion widths was higher. On smooth SiOx, the focal
adhesion widths were larger on average than on any of the patterned substrates
(Fig. 14). The variability in
focal adhesion widths was high on these substrates and very large loci of
vinculin staining seemed to arise from coalescence of multiple focal
adhesions. The measured widths of focal adhesions were larger than the ridge
widths on ridges up to 650 nm wide, but remained smaller than the pattern
pitches. On the 70 nm wide ridges on a 400 nm pitch, we occasionally observed
focal adhesions that were not aligned with the grooves and ridges and spanned
several grooves and ridges.
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Discussion |
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Human corneal epithelial cell alignment along micrometer- and
nanometer-sized grooves and ridges does not require the formation and
alignment of focal adhesions and stress fibers along the topographic features.
Focal adhesions and associated actin stress fibers were observed only
sporadically in aligned cells, after 12 hours of incubation, on all patterned
substrates. There are no previous reports on the orientation of focal
adhesions in epithelial cells cultured on topographically patterned
substrates. Focal adhesion and microfilament alignment was observed in
fibroblasts and macrophages aligned along micrometer-sized grooves and ridges
(Britland et al., 1996;
den Braber et al., 1998
;
Matsuzaka et al., 2000
;
Meyle et al., 1994
;
Walboomers et al., 1998
) and
was proposed to be the driving force for the observed contact guidance. The
generation of tension by actin stress fibers is known to be necessary for the
formation of focal adhesions
(Chrzanowska-Wodnicka and Burridge,
1996
). Nonetheless, fibroblasts have been reported to align along
grooves and ridges before well-formed actin filaments were observed
(Oakley and Brunette, 1993
;
Walboomers et al., 2000
).
Additionally, contact guidance, albeit delayed, has been observed to occur in
the presence of poisons that disrupt the actin cytoskeleton in fibroblasts
(Oakley and Brunette, 1995b
;
Oakley et al., 1997
;
Walboomers et al., 2000
;
Wojciak-Stothard et al.,
1995a
). We therefore conclude that focal adhesion and
microfilament alignment is not a prerequisite for contact guidance.
Our results do not support a mechanism for contact guidance solely on the
basis of the reaction of cells to surface discontinuities. The number of
discontinuities spanned by a single cell is ten times larger on substrates
with feature pitches of 400 nm than when the pitch is 2000 nm. However,
substrates with pitches ranging from 400 nm to 2000 nm yielded equivalent
percentages of aligned cells. Actin and vinculin condensations along surface
discontinuities have been described on macrophages
(Wojciak-Stothard et al.,
1995a), fibroblasts
(Wojciak-Stothard et al.,
1996
) and epithelial cells
(Oakley and Brunette, 1993
;
Oakley and Brunette, 1995a
)
cultured on micrometer-wide grooves and ridges. These were proposed to result
from the mechanical interaction of the cell membrane with the ridge edges and
were identified as the first step in the process of contact guidance in
fibroblasts (Wojciak-Stothard et al.,
1996
; Wojciak-Stothard et al.,
1995a
). Furthermore, actin condensations along substrate
discontinuities were still observed on cells treated with cytoskeletal
poisons, offering an explanation for the observed contact guidance on these
cells. We have seldom observed actin condensations along the ridge edges in
aligned cells. When we did observe actin condensations they were usually
continuous and located at the cell outline, unlike the condensations described
previously (Wojciak-Stothard et al.,
1996
; Wojciak-Stothard et al.,
1995a
), which had a punctuate morphology. These differences may be
ascribed to the use of different cell types. The percentage of aligned cells
only decreased when the pitch was 4000 nm, on substrates with 600 nm deep
grooves. Several studies report that the percentage of aligned cells is
inversely proportional to ridge width (den
Braber et al., 1996a
;
Walboomers et al., 1999a
). For
human corneal epithelial cells it appears that the ridge width of 1000 nm
(2000 nm pitch) is the threshold below which this effect is not detected, for
600 nm deep grooves.
Only a subpopulation of cells elongated and aligned along the patterned topographies. Many of the remaining cells appeared rounded up when observed using SEM (data not shown). Live cell imaging confirmed that a subpopulation of cells did not spread and remained round for the duration of the experiment (until 12 hours after cell seeding). Therefore, the low percentage of cells aligned along the patterned substrates reflects in part the inability of a subpopulation of human corneal epithelial cells to spread on the silicon oxide surface. Consistent with this, the percentage of nonspread cells (defined as cells with elongation less than 1.3 and area less than 600 µm2) was substantially larger on smooth or patterned silicon oxide surfaces than on TCPS (data not shown). In addition, the average areas of round cells were larger than the average areas of elongated cells (Figs 8 and 10). The chemical composition of the deposited layer of silicon oxide may therefore not be as favorable as TCPS for human corneal epithelial cell culture. Time-lapse microscopy also revealed that cells extended and retracted lamellipodia periodically and often acquired a round morphology after lamellipodial retraction, accounting for part of the population of round cells. By contrast, almost all NIH 3T3 fibroblasts cultured on similar patterned substrates aligned along the substrate features (data not shown). This is in agreement with the large percentages of fibroblasts that have been previously found to align along micrometer wide grooves and ridges.
Cell alignment was significantly greater on 600 nm deep than on 150 nm deep
grooves, for pitches ranging from 400 nm to 2 µm. Although not explored in
our studies, it is possible that the level of alignment we observed could be
further increased by increasing groove depth. An increase in cell alignment
with groove depth was observed in studies where the width of grooves and
ridges was in the micrometer range and the groove depths were 0.5 µm or
larger (Clark et al., 1990;
Walboomers et al., 1999b
). On
grooves with nanoscale depths, cell alignment also correlated with groove
depth, for both nanoscale (Clark et al.,
1991
) and micrometer-scale lateral feature dimensions
(Wojciak-Stothard et al.,
1996
). Cells have been found increasingly able to descend into the
grooves and form focal adhesions on the floor of the grooves as the ridge
width was increased and the groove depth decreased
(Walboomers et al., 1998
;
Walboomers et al., 1999a
). On
1 µm or 2 µm wide grooves, cells have rarely been reported to protrude
into the grooves (Walboomers et al.,
1999a
) and therefore most adhesions were observed to occur on the
ridge (Matsuzaka et al., 2000
;
Meyle et al., 1994
;
van Kooten and von Recum,
1999
). We observed that lamellipodia at the leading and trailing
edges of the cells did not descend into the grooves, for groove widths ranging
from 950 nm (2000 nm pitch) down to 330 nm (400 nm pitch), on both 150 nm and
600 nm deep grooves. On 2100 nm wide grooves lamellipodia frequently conformed
to the grooves on 150 nm deep grooves and were sometimes able to contact the
floor of the grooves on 600 nm deep grooves. Filopodia were able to adhere to
the grooves and ridges and were frequently aligned by the topographic
features. We therefore propose that filopodia play an important role in the
capacity of cells to perceive the depth of topographic features. Fibroblasts
were reported to produce more Fn mRNA on grooved substrates than on smooth
substrates (Chou et al., 1995
).
It was proposed that this response depends on groove depth and results from an
attempt to fill the grooves and stabilize the areas of the cell surface that
span the grooves (van Kooten and von
Recum, 1999
). However, thick protein deposits on the grooves have
not been observed in sections using transmission electron microscopy
(den Braber et al., 1998
), and
we have not detected them by cross sectional SEM. Unfavorable surface
chemistry for cell adhesion at the bottom of the grooves compared with the top
of the ridges has been proposed to account for the inability of cells to
contact the floor of the grooves
(Matsuzaka et al., 2000
;
Walboomers et al., 1999b
). We
averted this problem by coating the features with silicon oxide, obtaining a
uniform surface chemistry. An argument can be raised that differential
diffusion on the shallow and deep grooves accounts for the dependence of
alignment on groove depth. However, recent research suggests that increased
corneal epithelial cell migration on porous filters is independent of
diffusional limitations but attributable to the topographic features
themselves (Dalton et al.,
2001a
).
Cells cultured in the presence of serum aligned to the substrate
topographic patterns to a greater degree than cells cultured on the same basal
medium not supplemented with serum. Serum contains adhesive and nonadhesive
proteins that adsorb onto the surface before cells adhere. Therefore, the
chemistry of the surface that cells encounter during initial adhesion is
vastly different when serum is present from when no serum has been added to
the medium (Franco et al.,
2000). Formation of focal adhesions depends on the presence of ECM
proteins on the substrate (Burridge and
Chrzanowska-Wodnicka, 1996
). However, we found that some cells in
serum-free medium form focal adhesions after 12 hours of incubation,
suggesting that these cells deposited extracellular matrix proteins during
this period. Serum also provides cells with an array of growth factors that
initiate cell-signaling pathways on binding to cell-surface receptors. Some of
these pathways are interrelated to pathways stimulated by cell attachment to
the substrate and may therefore act in concert to amplify the effect of the
topographical stimuli. Interestingly, in the absence of serum, the average
cell area was similar on smooth silicon oxide and on TCPS. By contrast, the
average cell area in the presence of serum was approximately 50% larger on
TCPS than on silicon oxide. Supplementing the cell culture medium with serum
reduced the percentage of nonspread cells on TCPS by more than 50% compared to
serum-free medium but had no effect on the percentage of nonspread cells on
silicon oxide. This suggests that TCPS supports a layer of adsorbed protein
from the serum with more favorable compositions and/or conformations for cell
spreading than silicon oxide does.
Geometrical constraints imposed by the substrate dictated the focal
adhesion architecture. SEM revealed that the cell membrane bridges the
grooves, confining integrin occupancy to the top of the ridges. The width of
focal adhesions observed on cells cultured on substrates with ridge widths
larger than 400 nm was relatively constant. When the width of the ridges was
reduced to 70 nm, smaller than the reported width of mature focal adhesions
(250-500 nm) (Ohara and Buck,
1979) the average width of focal adhesions decreased. The measured
average focal adhesion width on 70 nm wide ridges (400 nm) is consistent with
the value expected when an object less than 100 nm wide is imaged by
fluorescence microscopy (Kozubek,
2001
). We therefore suggest that these focal adhesions are
confined to the top of one ridge. Interestingly, we have occasionally observed
focal adhesions that were not aligned with the topographic features and
spanned more than one pattern pitch. Aggregation of ligand-bound integrins on
these focal adhesions was not uniform but was restricted to the ridges.
Therefore, ligand spacings of at least 330 nm were presented to the cells.
Substrates with well-defined topographic features offer the opportunity to
investigate minimum ligand spacing and clustering required for the formation
of focal adhesions. They offer the advantage of providing well-defined
spacings and eliminating the possibility of haphazard ligand clustering
relative to smooth substrates coated with integrin binding ligands
(Maheshwari et al., 2000
;
Massia and Hubbell, 1991
).
Our results are consistent with a mechanism for contact guidance involving
anisotropic force generation by lamellipodia and filopodia. As described
above, we found that cells were able to align along the substrate features
without the alignment of focal adhesions and stress fibers. The formation of
focal adhesions and actin stress fibers is not necessary for cell spreading
(Cox and Huttenlocher, 1998;
Dunn and Brown, 1986
;
Greenwood and Murphy-Ullrich,
1998
). Cells must nevertheless exert a tractive force on the
substrate via focal complexes formed in filopodia and lamellipodia
(Dunn and Brown, 1986
). It has
previously been hypothesized that contact guidance results from filopodial
propagation being halted or hindered when filopodia face the ridge wall
(Dalton et al., 2001b
). We
found that filopodia that protruded from the sides of the cells were often
bent at the point of contact with the substrate and were subsequently aligned
with the grooves and ridges, preventing the generation of force in the
direction perpendicular to the ridges. Filopodia at the leading and trailing
edges of cells were often extended along the top of the ridges or the floor of
the grooves. Filopodial guidance by the substrate features was more prevalent
on the patterned topographies with the smaller pitches compared with the
micrometer-wide grooves and ridges. To bridge a groove, cell protrusions must
adhere to the top of a nearby ridge. The edges of lamellipodia were frequently
seen to extend into the grooves, especially on the patterns with 400 nm pitch.
Therefore, the failure of lamellipodia to reach adjacent ridges, becoming
effectively trapped in the groove, may contribute to inhibition of lateral
spreading. This effect was not observed on the widest and shallowest substrate
features, 150 nm deep grooves on a 4000 nm pitch, where lamellipodia were able
to conform to the topographic features. Consistent with this mechanism, live
cell imaging showed that protrusions perpendicular to the patterns were not
prohibited but were severely inhibited. Cell alignment resulted from
anisotropic cell spreading as cells extended and retracted lamellipodia
preferentially along the direction of the patterns.
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Acknowledgments |
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Footnotes |
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References |
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