An Experimental Model for Assessing
Fibroblast Migration in 3-D Collagen Matrices
Dimitris Karamichos, Neema Lakshman, and W. Matthew Petroll*
Department of Ophthalmology, University of Texas Southwestern Medical Center,
The purpose of this study was to develop and test a novel culture model for study-
ing fibroblast migration in 3-D collagen matrices. Human corneal fibroblasts were
seeded within dense, randomly oriented compressed collagen matrices. A 6 mm di-
ameter button of this cell-seeded matrix was placed in the middle of an acellular,
less dense outer collagen matrix. These constructs were cultured for 1, 3, 5 or 7
days in serum-free media, 10% fetal bovine serum (FBS), or 50 ng/ml PDGF. Con-
structs were then fixed and labeled with AlexaFluor 546 phalloidin (for f-actin)
and TOTO-3 (for nuclei). Cell-matrix interactions were assessed using a combina-
tion of fluorescent and reflected light confocal imaging. Human corneal fibroblasts
in serum-free media showed minimal migration into the outer (non-compressed)
matrix. In contrast, culture in serum or PDGF stimulated cell migration. Cell-
induced collagen matrix reorganization in the outer matrix could be directly visual-
ized using reflected light imaging, and was highest following culture in 10% FBS.
Cellular contraction in 10% FBS often led to alignment of cells parallel to the outer
edge of the inner matrix, similar to the pattern observed during corneal wound
healing following incisional surgery. Overall, this 3-D model allows the effects of
different culture conditions on cell migration and matrix remodeling to be assessed
simultaneously. In addition, the design allows for ECM density, geometry and me-
chanical constraints to be varied in a controlled fashion. These initial results dem-
onstrate differences in cell and matrix patterning during migration in response to
serum and PDGF. Cell Motil. Cytoskeleton 66: 1–9, 2009.
' 2008 Wiley-Liss, Inc.
Key words: extracellular matrix; corneal fibroblasts; cell migration; confocal microscopy; PDGF
Migration of activated corneal keratocytes (corneal
fibroblasts) plays an important role in matrix patterning
during developmental morphogenesis and is required for
repopulation of wounded corneal tissue following injury
or surgery [Jester et al., 1999; Netto et al., 2005]. In some
cases (e.g. following lacerating injury or incisional sur-
gery), contractile force generation is needed to facilitate
wound closure. In other circumstances (e.g. following re-
fractive surgery), it is preferable to have corneal fibro-
blasts repopulate the wound space without remodeling the
extracellular matrix or generating large forces, i.e. to
assume a regenerative migratory phenotype as apposed to
*Correspondence to: W. Matthew Petroll, Department of Ophthalmol-
ogy, Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas,
Texas 75390-9057, USA.
Contract grant sponsor: NIH; Contract grant numbers: EY013322,
EY016664; Contract grant sponsor: Research to Prevent Blindness,
Inc., NY (an Unrestricted Departmental grant and Senior Scientific
Investigator Award to WMP).
Received 27 August 2008; Accepted 5 November 2008
Published online 5 December 2008 in Wiley InterScience (www.
' 2008 Wiley-Liss, Inc.
Cell Motility and the Cytoskeleton 66: 1–9 (2009)
a contractile repair phenotype. Thus understanding how
cell-matrix mechanical interactions are regulated during
migration may facilitate the development of strategies to
modulate key aspects of corneal healing in vivo.
While there are standard models available for
measuring cell migration in 2-D culture, these models do
not simulate the 3-D environment cells encounter in
vivo. Furthermore, they do not allow assessment of cell-
induced matrix reorganization, a key event in wound
healing. In this study, we develop and test a novel exper-
imental model for assessing the pattern and amount of
cell migration within 3-D collagen matrices, as well as
the differences in local matrix patterning produced by
migrating cells in response to different culture condi-
tions. In this model, a compressed collagen matrix con-
taining cells (tissue equivalent) is placed within an acel-
lular, lower density outer matrix. The pattern and amount
of fibroblast migration from the inner to the outer matrix
is assessed using fluorescent labeling of cells, and cell-
induced matrix reorganization is evaluated using confo-
cal reflection imaging. Since the inner matrix is gener-
ated by external compression [Brown et al., 2005], the
cells are not required to be activated or contractile at the
start of the experiment. In addition, the matrix geometry,
cell numbers, and collagen density can be directly con-
trolled. Overall, this model may provide a unique and
flexible platform for investigating the migratory mechan-
ics of fibroblasts and other cells following exposure to
specific wound healing cytokines. Our initial results
demonstrate differences in cell and matrix patterning
during migration in response to serum (which activates
Rho) and PDGF (which activates Rac).
MATERIALS AND METHODS
A previously established, human corneal fibroblast
(HTK) extended lifespan cell line was used in this study
[Jester et al., 2003]. HTK cells were maintained in se-
rum-containing medium consisting of Dulbecco modi-
fied Eagle medium (DMEM; Gibco Invitrogen Cell Cul-
ture, Carlsbad, CA) supplemented with 1% penicillin/
streptomycin/fungizone (BioWhittaker Inc, Walkers-
ville, MD) and 10% fetal bovine serum (FBS; Sigma
Chemical, St. Louis, MD).
Preparation of Nested Collagen Matrices
Nested collagen constructs were prepared using
inner and outer matrices as described below.
Compressed Cellular Matrices (Inner matrices).
Rat tail type I collagen (10 ng/ml, BD Biosciences, San
Jose, CA) was diluted using Acetic acid to final concen-
tration of 3 mg/ml. Five milliliters of the mixture was
added to 0.5 ml of 103 MEM. After drop-wise neutrali-
zation with 1 M sodium hydroxide, a suspension of 1
million HTK cells in 0.5 ml DMEM was added to the
collagen mixture. The solution containing the cells and
the collagen was poured into a 3 3 2 3 1 cm3well, and
allowed to set for 1 h at 378C. Matrices were then com-
pacted by a combination of compression and blotting
using layers of mesh and paper sheets as previously
described (Fig. 1A) [Brown et al., 2005; Neel et al.,
2006]. Briefly, a 165 lm thick stainless steel mesh (mesh
size ? 300 lm) and a layer of nylon mesh (?50 lm
mesh size) were placed on a double layer of absorbent
paper. The constructs were placed on the nylon mesh,
covered with a second nylon mesh, and loaded with a
130 g stainless steel block for 5 min at room tempera-
ture, leading to the formation of flat collagen sheet
(?150 lm thick). A 6 mm diameter ‘‘button’’ was cut
out of this matrix using a trephine. In some experiments,
the button was then placed in 0.5% 5-(4,6-dichlorotria-
zinyl) aminofluorescein (DTAF) in 0.2 M NaHCO3 for
1 min to label the collagen. Pilot experiments demon-
strated that DTAF had no effect on cell viability. All
Cell-seeded collagen was placed between two nylon meshes and com-
pressed using a load for 5 min, during which time liquid was allowed to
exit through a stainless steel mesh at the bottom. B. Schematic of the
process for constructing the migration model. Cells were seeded in rat
tail collagen and compressed (as shown in A), and a 6 mm button was
punched out and placed inside an acellular uncompressed matrix.
A. Illustration of the collagen compression procedure is shown.
2Karamichos et al.
buttons were then washed in cell medium and placed
inside the acellular matrix, described below.
Acellular matrices (OUTER matrices). Rectan-
gular collagen constructs were prepared as described pre-
viously [Eastwood et al., 1998; Mudera et al., 2000; Kar-
amichos et al., 2007a]. Two milliliters of type I bovine
dermal collagen (3 mg/ml, PureCol. Inamed Corp, Fre-
mont, CA) was added to 0.2 ml of 103 MEM, neutral-
ized with 1M sodium hydroxide, and poured into 4.4 3
2.0 cm2Lab-Tek chambers (NUNC, Rochester, NY).
Bovine collagen was used to allow imaging of matrix or-
ganization using confocal reflection microscopy. The
compressed collagen button containing the cells was
then placed in the middle of this acellular matrix (Fig.
1B). Constructs were then allowed to set for 1 h at 378C.
Serum-free media (basal media) was prepared using
DMEM supplemented with 1% RPMI vitamin mix
(Sigma-Aldrich, Saint Louis, MO), 100 lM nonessential
amino acids (Invitrogen, Carlsbad, CA), 100 lg/mL
ascorbic acid, and 1% penicillin/streptomycin/fungizone.
Constructs were cultured in either basal media, basal
media supplemented with 50 ng/ml PDGF, or serum con-
taining media (10% FBS). Two different mechanical
constraints were used: (1) Attached (ATT) matrices in
which constructs were covered with medium after 1 h of
incubation (to allow polymerization of the outer matrix),
but remained attached to the bottom and sides of the
well, and (2) unconstrained (UN) matrices in which con-
structs were released from the bottom of the well after
polymerization, and allowed to float in medium.
F-Actin and DNA staining
At the end of each experiment (1, 3, 5, or 7 days)
constructs were fixed in 4% formaldehyde for 1 h and
permeabilized with 0.5% Triton X-100 in phosphate
buffer for 10 min. Cells were then incubated in phalloi-
din for 1 h (1:50 dilution, Alexa Fluor 546, Molecular
Probes, Eugene, OR) and then washed in PBS (3 times
for 5 min). TOTO-3 iodide (1:1000; Molecular Probes)
was then added to each construct to stain the cell nuclei.
Constructs were incubated for 15 min and washed with
PBS (3 times for 5 min).
Laser Confocal Microscopy
After staining, buttons were mounted onto 25 lm
thick, 60 mm diameter, mylar petri dishes (Bachofer,
Hamburg,Germany) for imaging. Fluorescent imaging
was used for DTAF, F-actin and TOTO-3 whereas
reflected light was used for collagen fibril imaging. Se-
quential scanning was used to prevent crosstalk between
fluorophores. 3-D optical stacks (z-series) were acquired
using confocal microscopy using both a 203 dry objec-
tive (2 lm steps) and a 633 water immersion objective
(1 lm steps). Overlapping stacks of images were
collected using the 203 objective, from the edge of the
inner matrix until the last cell was reached (Fig. 2). This
was repeated for up to four different quadrants (north,
east, south, west) for each construct. The use of the
mylar Petri dishes and the fact that the inner matrices
were placed near the bottom of the outer matrices (<
500 lm) insured that there was sufficient working dis-
tance to reach all of the migrating cells with the 203
objective in most cases.
Maximum intensity projection (MIP) images of
phalloidin and TOTO-3 image stacks were created using
Metamorph. Photoshop was then used to align overlap-
ping MIP images, resulting in a 750 lm wide montage
image for each quadrant. These images were used to
measure the distance of the leading edge cells from the
inner-outer matrix interface. These were the cells that
had traveled the farthest from the interface; a straight
line between the leading cell end and the interface was
used for this distance measurement. To assess cell-
induced matrix reorganization, 633 image stacks were
collected both near the edge of the inner matrix, and at
the leading edge of the migratory front.
Statistical analyses were performed using Sigma-
stat (version 3.1.1; Systat Software Inc., Point Richmont,
CA). ANOVA was used to compare group means and
differences were considered significant when P < 0.05.
Effect of Culture Conditions on the Pattern and
Amount of Cell Migration
As a model for assessing cell migration, corneal
fibroblasts were embedded in a compressed inner matrix,
in each sample using laser confocal microscopy.
Schematic showing the pattern of 3-D image stacks collected
Fibroblast Migration in 3-D Matrices3
which was placed within in an uncompressed acellular
outer matrix. At 24 h, cells were observed at the edge of
the inner matrix with only occasional pseudopodia par-
tially extending into the outer matrix. Migration of entire
cells into the outer matrix was not generally observed
until 2–3 days after plating these constructs (Figs. 3A–
3C). The edge of the inner matrix was easily discernable
using confocal reflection imaging, as confirmed by using
DTAF to label the collagen in the inner matrix. In some
cases, the edge of the inner matrix was folded, and/or a
gap was detected between the inner and outer matrices
(these samples were excluded); however, in most cases
the mechanical integrity of the constructs was main-
By 5 days, there were striking differences in the
number of cells in the outer matrix, as well as the dis-
tance they traveled under different culture conditions
(Figs. 3D–3F). Corneal fibroblasts in serum-free media
showed minimal migration into the outer matrix. In con-
trast, culture in serum or PDGF stimulated cell migra-
tion. This increase was even more pronounced after 7
days (Fig. 4). These responses were observed in both the
ATT and UN models.
We often observed cells aligned parallel with the
outer edge of the inner matrix following culture in 10%
FBS. This alignment was first observed at 5 days (Fig.
3E, arrows) and was more pronounced at 7 days (Figs.
4B and 4C, arrows) in both ATT and UN models. These
cells were on the outside edge of the inner matrix; thus
they had already migrated into the uncompressed outer
matrix. In contrast, we did not observe any consistent
pattern of alignment of cells within the inner compressed
into the outer matrix. A–C: DTAF and TOTO-3 overlays collected af-
ter 3 days of culture in basal media (A), 10% FBS (B), and PDGF (C).
D–F: Reflected light and phalloidin overlays collected after 5 days of
Maximum intensity projection images showing cell migration
culture in basal media (D), 10% FBS (E), and PDGF (F). Both serum
and PDGF stimulated cell migration from the inner (left) into the outer
(right) matrix. Note the alignment of cells along the inner-outer matrix
interface after 5 days of culture in 10% FBS (E, arrows).
4 Karamichos et al.
matrix. In order to determine whether this effect was
dependent on serum concentration, we performed 7 day
experiments using 1% FBS. One percent of FBS stimu-
lated cell migration (Fig. 4E), but most cells remained
oriented perpendicular to the edge of the inner matrix,
similar to PDGF.
Figure 5 shows the average distance of the leading
edge of cell migration from the edge of the inner matrix
after 7 days of migration. Cells cultured in 1% FBS,
10% FBS, and PDGF migrated significantly farther (P ?
0.05) than those in basal media.
Matrix Reorganization Produced
During Cell Migration
Local cell-matrix interactions were assessed using
high magnification confocal reflection imaging at two
different sites within the constructs: (1) At the interface
between the inner and outer matrices, and (2) at the lead-
ing edge of the migration front. Figure 6 shows the cell-
matrix interactions for all three conditions after 3 days in
culture. Morphologically, cells under all conditions were
generally bipolar and had pseudopodial processes, char-
acteristics typical of corneal fibroblasts in 3-D matrices.
At the interface between the inner and outer matrices,
some compaction and alignment of ECM was observed
under all conditions studied. However, the largest
amount of cell-induced collagen reorganization was con-
sistently observed following culture in media containing
10% FBS (Fig. 6B, arrows), as compared to basal media
jection images collected after 7
days of culture in basal media (A),
10% FBS (B and C), PDGF (D),
and 1% FBS (E). Both serum and
PDGF stimulated cell migration
from the inner (left) into the outer
(right) matrix. Note the dramatic
alignment of cells along the inner-
outer matrix interface following
culture in 10%FBS (B and C,
arrows), but not 1% FBS or PDGF.
Maximum intensity pro-
outer matrix under different culture conditions. The distances were
measured from the interface to the leading edge of the migrating front.
Quantitative analysis of the distance cells traveled into the
Fibroblast Migration in 3-D Matrices5
(Fig. 6A) and media containing PDGF (Fig. 6C). Under
all conditions, increased matrix alignment was often
observed between neighboring cells (Fig. 6C, double
arrow), suggesting cell-cell mechanical enhancement of
ECM reorganization. Cell-induced matrix organization
was also analyzed at the furthest point from the inner
matrix, i.e. at the leading edge. Increased matrix reorgan-
ization was again observed following culture in 10%
FBS (Fig. 6E, arrows), as compared with culture in basal
media or PDGF. By 5 days, the matrix was often com-
pacted to a point where individual collagen fibrils could
not be easily distinguished using confocal reflection
imaging, particularly under 10% FBS conditions.
Visual inspection of the unconstrained constructs
revealed that following 5–7 days of culture in 10% FBS
the outer matrix was often contracted and reduced in size
along both the x and y axes. In contrast matrix size
reduction was not observed following culture in basal
media or PDGF. In the attached constructs, cells were
often able to pull the matrix from the sides of the wall
across the x-axis after 7 days of culture in 10% FBS, and
create a visible ‘‘waist’’. These samples were sometimes
wrinkled and distorted along the bottom, suggesting
partial detachment along this plane as well.
Stromal keratocytes play a central role in media-
ting the corneal response to lacerating injury or refrac-
tive surgery [Netto et al., 2005]. During wound healing,
quiescent corneal keratocytes differentiate into fibroblast
and/or myofibroblast phenotypes that mediate cell migra-
tion, wound contraction and matrix remodeling [Jester
et al., 1999]. While there are standard models available
for measuring the effects of growth factors on the rate of
cell migration in 2-D culture, models for assessing cell
migration within 3-D matrices are more limited. One
approach is to embed tissue explants within acellular col-
lagen matrices, and study cells as they migrate out
[Stopak and Harris, 1982; Sawhney and Howard, 2002].
and DTAF (blue), and confocal reflection images of collagen fibrils
(red) following 3 days of culture in basal media (A and D), 10% FBS
(B and E) and PDGF (C and F). A–C: At the interface between the
inner and outer matrices, some compaction and alignment of ECM
was observed under all conditions studied. However, the largest
amount of cell-induced collagen reorganization was consistently
observed following culture in media containing 10% FBS (B, arrows).
Color overlays of fluorescent images of phalloidin (green)
D–F: Cells located near the furthest point from the inner matrix (i.e.,
the leading edge). Increased matrix reorganization was again observed
following culture in 10% FBS constructs (E, arrows), as compared
with culture in basal media or PDGF. Under all conditions, increased
matrix alignment was often observed between neighboring cells (for
example C, double arrow), suggesting cell-cell mechanical enhance-
ment of ECM reorganization.
6 Karamichos et al.
However, the corneal stroma is maintained in a highly
dehydrated state in vivo, and swells dramatically when
explanted; thus this technique is problematic when using
corneal explants and is highly dependent upon the source
of the tissue. Another approach is to place cells on the
surface of collagen matrices and measure their move-
ment into the interior [Schor, 1980; Andresen et al.,
2000, 2007; Sabeth et al., 2004], but this does not simu-
late the 3-D geometry of wound healing.
More recently, Grinnell and coworkers used a
nested matrix approach for studying 3-D cell migration
[Greiling and Clark, 1997]. In this model, human fore-
skin fibroblasts are stimulated to contract a collagen ma-
trix, and this pre-contracted matrix is placed within a
second acellular collagen matrix [Grinnell et al., 2006].
Fibroblast migration into the outer matrix is then fol-
lowed over time. This elegant model has provided novel
insights into the regulation of dermal fibroblast migra-
tion by specific cytokines and mechanical signals [Grin-
nell et al., 2006; Jiang et al., 2008; Miron-Mendoza
et al., 2008]. However, since our ultimate goal is to study
the transformation and migratory response of quiescent
(i.e. non-contractile) corneal keratocytes to specific
wound healing cytokines, we needed a model that did
not require cellular pre-contraction of the inner matrix.
The compressed collagen matrix model was selected
because collagen is compacted by using external com-
pression [Brown et al., 2005], thus the cells are not
required to be contractile or activated at the start of the
experiment. Furthermore, compressed matrices support
differentiation of both activated fibroblasts and quiescent
keratocytes (unpublished observation), and have a colla-
gen concentration and mechanical stiffness which are
similar to that of native corneal tissue [Neel et al., 2006].
We also used a cell density in the compressed matrices
(?11,000 cells/mm3) that is of the same order as that in
the human corneal stroma in vivo [Patel et al., 2001].
Thus in this study, the compressed inner matrices were
engineered to simulate key properties of in vivo corneal
tissue. However, a key feature of our approach is that the
cell density, geometry, and collagen density (of both the
inner and outer matrices) can all be modulated during
preparation in order to mimic the properties of other
In our model, cell migration into the outer matrix
was not generally observed until 2–3 days after plating
the constructs. During this lag phase, cells were observed
at the edge of the inner matrix with only occasional
pseudopodia partially extending into the outer matrix. It
is possible that this delay is due to the large difference in
mechanical stiffness between the inner and outer matri-
ces, since cells generally prefer to migrate from soft to
stiff environments [Lo et al., 2000]. It may also be
related to the high density of collagen within the com-
pacted inner matrix, through which the cells must
migrate in order to reach the outer matrix. While the ori-
gins of this delay are currently unclear, it should be
noted that a similar lag phase is observed during corneal
wound healing in vivo, in which cells also migrate out of
a mechanically stiff ECM [Jester et al., 1995, 1999]. Fur-
ther studies in which the cell and/or collagen density are
varied in our model may clarify our understanding of
this phenomenon. One advantage nesting a pre-con-
tracted matrix is that there is a much shorter lag phase
(8–16 h) than in our model [Grinnell et al., 2006]. This
may be due to the activated state of the cells following
contraction, or differences in overall matrix geometry
and mechanical properties.
By 5 days, we found striking differences in the
number of cells in the outer matrix as well as the dis-
tance they traveled under different culture conditions.
Both serum and PDGF stimulated significant cell migra-
tion into the outer matrix, and this was even more pro-
nounced after 7 days. PDGF is expressed in the human
cornea, and stimulates migration of corneal fibroblasts
plated on rigid substrates or on top of collagen matrices
[Kim et al., 1999; Andresen et al., 2000, 2007]. In 3-D
culture, PDGF activates Rac and stimulates corneal
fibroblast spreading in single collagen matrices [Petroll
et al., 2008a], as well as dermal fibroblast migration in
nested collagen matrices [Grinnell et al., 2006]. PDGF
also stimulates proliferation of corneal fibroblasts [Kim
et al., 1999], thus the number of cells observed in the
outer matrix at 5 and 7 days likely reflects a combination
of both migratory and proliferative responses. Prolifera-
tion of corneal fibroblasts also occurs during in vivo
wound healing [Jester et al., 1999; Netto et al., 2005].
While significant migration was also observed fol-
lowing culture in 10% FBS, important differences in the
pattern of cell alignment were observed. Cells in 10%
FBS tended to align parallel to the outer edge of the
inner matrix beginning at 5 days; whereas cells cultured
in PDGF were aligned perpendicular to the edge of the
inner matrix. This alignment was even more pronounced
after 7 days. Interestingly, a similar pattern of cell align-
ment is observed during corneal wound contraction fol-
lowing incisional surgery [Petroll et al., 1993; Petroll
et al., 1998]; in these studies a force-balance analysis
predicted that as cells generate contractile forces they
pull themselves into alignment with the wound edge, due
to its increased rigidity as compared to the ECM within
the wound space. While serum contains several pro-mi-
gratory growth factors (including PDGF), it also contains
factors such as lysophosphatidic acid (LPA) and sphin-
gosine-1-phosphate (S1P) which stimulate cell contrac-
tility through activation of the Rho/Rho Kinase pathway
[Grinnell, 2000; Jiang et al., 2008; Petroll et al., 2008b].
Thus the shift in cell alignment observed at 5 and 7 days
Fibroblast Migration in 3-D Matrices7
may reflect a transition from a Rac-induced migratory
phenotype to a Rho-induced contractile phenotype. In
order to determine whether this effect was dependent on
serum concentration, we performed 7 day experiments
using 1% FBS. One percent of FBS stimulated cell
migration, but most cells remained oriented perpendicu-
lar to the edge of the inner matrix, similar to PDGF.
Thus the balance between Rho and Rac activation
appears to change as the serum concentration is
increased, with Rho predominating at higher concentra-
To directly assess cell-induced matrix reorganiza-
tion during migration, confocal reflection imaging was
used [Friedl et al., 1997; Friedl and Brocker, 2000].
After 3 days, some compaction and alignment of ECM
was observed under all conditions studied. However, the
largest amount of cell-induced collagen reorganization
was consistently observed following culture in media
containing 10% FBS, as compared to basal media or
PDGF. The residual matrix reorganization observed fol-
lowing culture in serum-free media or PDGF is most
likely due to a basal level of Rho-kinase activity in this
corneal fibroblast cell line [Petroll et al., 2008a], that
would not be expected in quiescent corneal keratocytes
[Jester and Chang, 2003].
It is important to note that visual inspection of the
unconstrained constructs revealed that following 5–7
days of culture in 10% FBS the outer matrix was often
contracted and reduced in size. In contrast matrix size
reduction was not observed following culture in basal
media or PDGF. In the attached constructs, cells were of-
ten able to detach the matrix from the sides of the wall
across the x-axis after 7 days of culture in 10% FBS, and
create a visual ‘‘waist’’. A similar waist has been
reported in partially restrained matrices using contractile
cells [Brown et al., 1998; Eastwood et al., 1998; Karami-
chos et al., 2007b].
Taken together, our results indicate that there is
increased cell contractility and cell-induced matrix reor-
ganization during migration in the presence of 10% FBS
as compared to culture in PDGF. Thus PDGF may be a
candidate for stimulating wound healing following re-
fractive surgery in vivo, where it is preferable to have
corneal fibroblasts repopulate the wound space without
remodeling the extracellular matrix or generating large
contractile forces; both of which can alter corneal clarity
and/or refractive power.
In nested collagen matrices, dramatic differences
in dermal fibroblast migration have been observed
between ATT and UN conditions [Miron-Mendoza et al.,
2008]. In the current study, however, a similar pattern
and amount of cell migration was observed under
both conditions. The reason behind this is not entirely
clear, but is likely related to the fact that the distance
from matrix attachment points to the edge of the inner
matrix was relatively large in our model, and thus the
constraints may have little impact on the effective
mechanical stiffness initially encountered by the cells.
Furthermore, the collagen density of the outer matrix
was higher in the current study (2.5 mg/ml vs. 1.5 mg/
ml), providing increased rigidity even under UN
conditions. Further investigation is clearly required to
clarify the role of these and other variables on the
mechanics of cell migration by varying these parameters
in our model.
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