Biomaterials 35 (2014) 1496e1506

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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Regulation of migratory activity of human keratinocytes by

topography of multiscale collagen-containing nanobrous matrices
Xiaoling Fu a, b, c, Meng Xu a, Jie Liu d, Yanmei Qi d, Shaohua Li d, Hongjun Wang a, *
a

Department of Chemistry, Chemical Biology and Biomedical Engineering, Stevens Institute of Technology, Hoboken, NJ 07030, USA
The National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510640, Peoples Republic of China
c
The School of Materials Science and Engineering, South China University of Technology, Guangzhou 510640, Peoples Republic of China
d
Department of Surgery, Robert Wood Johnson Medical School, New Brunswick, NJ 08903, USA
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 8 October 2013
Accepted 2 November 2013
Available online 20 November 2013

Nanobrous matrices hold great promise in skin wound repair partially due to their capability of
recapturing the essential attributes of native extracellular matrix (ECM). With regard to limited studies
on the effect of nanobrous matrices on keratinocytes, the present study was aimed to understand how
the topographical feature of nanobrous matrices regulates keratinocyte motility by culturing keratinocytes on polycaprolactone (PCL)/collagen nanobrous matrices (rough surface with ber diameters of
331
112 nm) or the matrices coated with a thin layer of collagen gel to form a secondary ultrane
brous network (smooth surface with ultrane ber diameters of 55
26 nm). It was found that the PCL/
collagen nanobrous matrices alone did not stimulate cell migration, while collagen gel coating could
signicantly increase cell motility. Further studies demonstrated that the ultrane brous network of
collagen gel coating signicantly activated integrin b1, Rac1 and Cdc42, facilitated the deposition of
laminin-332 (formerly called laminin-5), and promoted the expression of active matrix metalloproteinases (MMPs) (i.e., MMP-2 and 9). Neutralization of integrin b1 activity abrogated the gel
coating-induced keratinocyte migration. These ndings provide important evidence on the role of
topographical features of nanobrous matrices in regulating the phenotypic alteration of keratinocytes
and suggest the possible utility of collagen-containing nanobrous matrices for skin regeneration
especially in re-epithelialization.
2013 Elsevier Ltd. All rights reserved.

Keywords:
Nanobers
Human keratinocytes
Topography
Reepithelialization
Integrin b1
Laminin-332 (laminin-5)

1. Introduction
In skin wound repair, rapid restoration of the epithelial barrier
function is crucial to minimize potential threats from the environment such as bacterial invasion and dehydration [1]. Extensive
efforts have been made to regenerate epidermis by transplanting
cultured epithelial cell sheets [2e5], grafting meshed split skin
[6,7], using tissue-engineered skin grafts with an epidermal layer
[8e10], or stimulating the re-epithelialization from wound edge or
intact hair follicles [1,11]. In either approach, complete reepithelialization cannot be achieved until further migration of
keratinocytes from the graft and wound edge to bridge the open
wound surface.

* Corresponding author. Department of Chemistry, Chemical Biology and

Biomedical Engineering, Stevens Institute of Technology, McLean Building Room
416, Castle Point on Hudson, Hoboken, NJ 07030, USA. Tel.: 1 201 216 5556;
fax: 1 201 216 8240.
E-mail address: Hongjun.Wang@stevens.edu (H. Wang).
0142-9612/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biomaterials.2013.11.013

In recognition of the importance of keratinocyte migration in

wound re-epithelialization, it is highly desirable to create a
microenvironment with the ability to promptly recruit autologous
keratinocytes from the intact wound edge and promote their
migration for faster re-epithelialization. Electrospun nanobrous
matrices, featuring high surface-to-volume ratio, high porosity and
good interconnectivity, have great potential to mimic the skin ECM
in both morphology and composition [12,13]. It has been reported
that nanobrous matrices support the adhesion and spreading of
normal human keratinocytes [14]. However, the most critical
behavior of keratinocyte in wound healing, i.e., migration on electrospun nanobrous matrices has never been studied.
The activation of keratinocyte migration can initiate soon after
injury, while its migration rate signicantly relies on the wound
bed and underlying ECM [15e17] and involves the synergistic coordination among several cellular events including cell attachment/
detachment, cytoskeletal reorganization, ECM degradation and
redistribution of membrane integrins [16,18,19]. It has been found
that ECM molecules like collagen type I and type IV, laminin,
bronectin and vitronectin can regulate keratinocyte migration by

X. Fu et al. / Biomaterials 35 (2014) 1496e1506

interacting with various integrins on the cell surface [16,17,20e22].

In this regard, a plausible strategy for creating skin grafts is to
incorporate relevant ECM molecules in the scaffolds, for example,
either directly incorporated into electrospun nanobers or coated
onto the ber surface post-electrospinning [23], to promote keratinocyte proliferation and migration. Furthermore, transplantation
of such grafts to the wound bed can also formulate a stimulatory
microenvironment to recruit autologous keratinocytes for an
accelerated re-epithelialization. Upon injury, keratinocytes of the
epidermis are activated and migrate onto dermis, which is rich with
type I collagen brils [16,17,24], suggesting the stimulatory effect of
type I collagen on keratinocyte migration. Previous results showed
that type I collagen-containing electrospun nanobers promote the
adhesion and spreading of human epidermal keratinocytes and
support the formation of epidermal layers [25]. However, electrospun collagen bers normally have diameters ranging from 100 to
1200 nm [25,26], much larger than collagen brils in dermis
(w50 nm in diameter), which would result in different surface
roughness and subsequently induce differential keratinocyte
migratory response. In this regard, it becomes necessary to determine whether electrospun nanobers are optimal for wound repair,
especially for rapid re-epithelialization, and if not, what kind of
modication should be made to further improve them.
In this study, collagen-containing nanobrous matrices with
two different topographical features, i.e., electrospun polycaprolactone (PCL)/collagen nanobrous matrices with an average
ber diameter of 331
112 nm and electrospun PCL/collagen
nanobrous matrices coated with a thin layer of type I collagen gel
to form a secondary ultrane brous network (average diameter of
55
26 nm) bridging the electrospun bers, were fabricated. The
proliferation, morphology and migration of human epidermal
keratinocytes on both nanobrous matrices were side-by-side
studied.
2. Materials and methods
2.1. Materials
1,1,1,3,3,3-hexauoro-2-propanol (HFIP) was obtained from Oakwood products
Inc (West Columbia, SC). Poly(epsilon-caprolactone) (PCL, Mw 80,000) was purchased from SigmaeAldrich (St. Louis, MO) and type I collagen (Coll) was obtained
from Elastin Products Inc. (Owensville, MO). All other reagents and solutions were
obtained from Invitrogen (Carlsbad, CA) except as indicated.
2.2. Nanobrous matrix preparation, modication and characterization
Nanobrous matrices of collagen or PCL/Coll were fabricated using the electrospinning technique following the same procedures as previously described [27].
Briey, 8% (w/v) pure collagen or 3:1 PCL/Coll (w/w) blend solutions were prepared
by dissolving PCL and collagen in HFIP. Then, the solution was loaded into a 5-mL
syringe with a 20-gauge blunt-tip stainless steel needle and electrospun at 15 kV
using a customized electrospinning apparatus. The polymer solution was dispensed
using a syringe pump (Kdscientic, Holliston, MA) at 10 mL/min. Nanobrous
matrices collected onto round glass cover slips ( 12 mm) under sterile conditions
were used for the studies unless indicated. Then, PCL/Coll nanobrous matrices
were placed in 24-well culture plates.
To modify the electrospun nanobrous matrices with collagen gel, fresh collagen
solution was prepared by dissolving type I collagen in 0.01 N HCl and stored at 4  C
for further use. To modify the PCL/Coll nanobrous matrices, prepared collagen
solution was diluted in PBS at a ratio of 4:1 and then neutralized by adding 0.1 N
NaOH on ice. The neutralized collagen solution with a nal concentration of 3 mg/
mL was added onto the PCL/Coll nanobrous matrix surface and then immediately
removed as much as possible. After incubation for 1 h at 37  C for a complete
gelation, the matrices were washed with PBS and then immediately used for cell
culture or for characterization as described below. To better compare the effect of
substrate surface on keratinocyte behavior, two additional groups were also
included: collagen adsorbed PCL/Coll nanobrous matrices and electrospun collagen
nanobrous matrices. For the former, a neutralized collagen solution of 50 mg/mL
was added onto PCL/Coll nanobrous matrix surface and incubated for 2 h at room
temperature for adsorption. After adsorption, the meshes were washed with PBS
and used for cell culture or further characterization.
Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were
used to characterize these nanobrous matrices. For SEM examination, the

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dehydrated samples were sputter-coated with gold and then examined with a LEO
982 FEG SEM. To determine the diameter of nanobers, images of ve randomly
selected areas were captured and analyzed by analysis software (NIS-elements BR
3.10, Nikon, Japan). The surface roughness of different nanobrous matrices was
determined by using Agilent NanoR AFM in a dynamic force mode, that is, a
tapping mode. Three randomly selected areas of the surface with the size of
10  10 mm (x, y direction) were scanned, respectively. Close contact mounted
cantilevers (Pacic Nanotechnology, Santa Clara, CA) were used. To describe the
topography and roughness of the substrates, the roughness parameter for the surface, Rms, the root-mean-square height of the surface, was calculated by NanoRule.
2.3. Cell and cell culture
Human keratinocytes immortalized by expressing the catalytic subunit of telomerase were a gift from Dr. James Rheinwald (NIH Harvard Skin Disease Research
Center). Cells were plated in serum-free keratinocyte medium supplemented with
recombinant EGF (2.5 mg/500 mL), bovine pituitary extract (25 mg/500 mL), 0.3 mM
CaCl2, 100 mg/mL streptomycin and 100 IU/mL of penicillin (Sigma, St Louis, MO) and
cultured at 37  C in a humidied 5% CO2 atmosphere. Medium was replaced every
48 h until they reached 50e60% conuence.
Keratinocytes were seeded onto various nanobrous matrices or glass cover
slips at a density of 5  103 cells/cm2 and allowed to attach for 60 min prior to the
addition of fully supplemented medium. The cells were further cultured for up to 7
days. The effect of nanobrous matrices on cell behaviors was determined
throughout the 1-week culture period.
2.4. Cell migration assay
In vitro CytoSelect 24-Well Wound Healing Assay Kit purchased from Cell
Biolabs Inc. (San Diego, CA) was used to evaluate the migratory capacity of keratinocytes. Briey, 2  105 keratinocytes suspended in the culture media were seeded
onto various matrix surfaces with inserts in place. After culturing for 12 h, the insert
was removed to generate a consistent 0.9 mm wound gap among the cells. Cells
were allowed to migrate into the wound gap for 24 and 48 h. After staining the cells
with methylene blue, images of the wound gap were taken to analyze the migration
distance of keratinocytes. 10 representative points along the wound of each
sample were used to evaluate average migration (n 4).
To determine the involvement of integrin b1 in regulating keratinocyte migration, anti-integrin b1 antibody (Millipore, Clone P4C10, 1:200) was added to the
culture right after the removal of insert to neutralize the integrin b1 function.
2.5. Cell proliferation
Cell proliferation on various substrates was measured using CyQUANT Cell
Proliferation Assay Kit (Molecular Probes, Inc., Eugene, OR) following the protocol
suggested by the manufacturer. Briey, A standard curve over cell number was rst
established (y 0.007x  2.977, R2 0.995). Samples were then harvested on day 1,
3 and 7 (n 4 per group for each time point). After removal of medium and washing
with PBS, the samples were frozen and stored at 80  C. After collecting all the timepoint samples, the samples were lysed in CyQUANT cell-lysis buffer for 1 h at room
temperature and then 200 mL of CyQUANT GR dye/cell-lysis buffer was added to
each sample and incubated for 2e5 min in the dark at room temperature. The
uorescence of cell lysates was measured using the multi-mode BioTek microplate
reader at w480 nm excitation and w520 nm emission. Cell numbers were calculated
based on the standard curve.
2.6. Immunouorescent microscopy
Immunouorescent staining of cells was performed following the procedures
previously described [28]. Briey, cultured cells were xed in 4% paraformaldehyde
for 10 min and then permeabilized with 0.2% Triton X-100 in PBS. Antibodies used in
the study were as follows: phalloidin-TRITC (Sigma, 1:500), anti-vinculin-FITC
(Sigma, 1:200), antibody against the active conformation of b1 integrin (Millipore,
clone HUTS-4, 1:300) and anti-laminin-332 (also known as anti-laminin-5, Millipore, clone P3H9-2, 1:200). For those primary antibodies without uorescent labels,
the cells were further incubated with goat anti-mouse IgG-FITC conjugate secondary
antibody (Caltag, 1:400). Cell nuclei were stained with DAPI (Sigma, 1:1000). The
staining was examined under a Nikon Eclipse 80i epiuorescent microscope. The
uorescence intensity of the lysate of cells stained for active integrin b1 was also
quantied by using a multi-mode microplate reader (Synergy HT, BioTek Instruments, Inc., Winooski, VT).
2.7. Determination of polarized keratinocytes
Keratinocytes were seeded on various matrices at a density of 2  104 cells/cm2
and cultured for 24 h and then xed with 4% formaldehyde. After staining the cells
with TRITC-phalloidin and DAPI, images were taken from three randomly selected
elds containing approximately 200 cells for each culture well. To score a cell as
polarized, the cell needed to possess two dened morphological features, including
(1) elongation and (2) the presence of long cytoplasmic extensions (lamellipodia and

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X. Fu et al. / Biomaterials 35 (2014) 1496e1506

lopodia). Cells that did not show these two features but spread were counted as
nonpolarized. Cells that were not spread were not counted.
2.8. Zymography
The expression of MMP-2 and MMP-9 in keratinocytes was analyzed by
zymography as described previously [29,30]. Briey, supernatants of each culture
were collected on day 1, 3 and 7 and stored at 80  C for further analysis. The protein
concentration of each sample was determined by bicinchoninic acid assay (BCA
protein assay kit; Pierce, Rockford, IL). Equal amounts of proteins (5 mg) were loaded
on 10% sodium dodecyl sulfate (SDS)epolyacrylamide gels (Sigma) containing 1%
gelatin (Sigma) and then separated by electrophoresis on ice (at 125 V for 120 min).
The proteins were renatured in the renaturing buffer (Biorad, Hercules, CA) at room
temperature for 1 h, allowing gelatinases to cleave the gelatin substrate copolymerized in the gel. The gel was then incubated with developing buffer (Biorad) at
37  C overnight. Afterwards, the gel was stained with 0.5% Coomassie blue for
30 min, and then destained with distilled water for appropriate color contrast. Clear
bands were indicative of the location of proteolytic activity. The identities of the
MMPs were based on their molecular weights.
2.9. Measurement of the activity of integrin b1 by ow cytometry
Analysis of cell surface expression of active integrin b1 by ow cytometry was
performed as described previously [31]. 1  106 broblasts seeded on various
matrices were trypsinized and washed with FACS buffer (5% BSA, 0.1% sodium azide
in PBS). Cells were resuspended and incubated with 2.5 mg HUTS-4 or isotypematched IgG (IgG2b) negative control (Millipore) for 1 h at 4  C. After washing
with the FACS buffer, the cells were labeled with FITC-conjugated secondary antibody for 1 h at 4  C. Cells were then washed and analyzed with the Cell Lab Quanta
SC MPL ow cytometer using Quanta analysis program (Beckman Coulter, USA).
Approximately 5000 cells were analyzed in each experiment, and the results shown
were representative of three independent experiments.
2.10. Measurement of Rac1 and Cdc42 activation by afnity pull-down assay
The activation of Rac1 and Cdc42 was measured based on the specic interaction
of activated Rac1 and Cdc42 with their effectors PAK1 p21-binding domain (PBD)
and N-WASP GTPase binding domain (GBD), respectively [32]. Briey, PBD and GBD
cDNAs in the pGEX-2T plasmid were expressed in Escherichia coli, and bacteria were
lysed by sonication. The PBD-GST and GBD-GST fusion proteins were isolated with
glutathione-agarose beads (GE Healthcare Life Sciences). Cell lysates containing
1 mg of proteins were incubated with 15 mg of the bead-bound GST fusion proteins at
4  C for 60 min. Afnity-puried proteins were separated by 12% SDS-PAGE,
transferred to polyvinylidene diuoride (PVDF) membranes, and immunoblotted
using Rac1 and Cdc42 antibodies. Bound primary antibody was detected with antimouse horseradish peroxidase (HRP)-conjugated secondary antibody and chemiluminescence detection reagents. The bands were assessed by densitometry (Image
J), and the amount of PBD-bound Rac1 and GBD-bound Cdc42 was compared with
that of total cellular Rac1 and Cdc42 determined by direct immunoblot analysis.
2.11. Statistical analysis
Each experiment was repeated at least 3 times on different time points and data
were expressed as the mean
SD. All the cytotoxicity and cell attachment measurements were collected in triplicate for each group. Unpaired student t-test was
used to evaluate the signicance between experimental groups. A value of p < 0.05
was considered statistically signicant.

3. Results
3.1. Characterization of nanobrous matrices
Using the established electrospinning condition as described in
Materials and Methods, electrospun PCL/Coll nanobers collected
on the glass cover slips had a random arrangement with an average
diameter of 331
112 nm (Fig. 1A and Table 1). Distinctively, the
coating of PCL/Coll electrospun nanobrous matrices with 3 mg/mL
collagen gel yielded a secondary ber network (arrowhead in
Fig. 1B and Table 1) with an average diameter of 55
26 nm. These
ultrane bers of collagen gel evenly distributed among the
interstitial space of PCL/Coll nanobers (asterisks in Fig. 1B) in a
random fashion to bridge the PCL/Coll nanobers and formed a
smoother surface. Close examination of these ultrane bers
revealed the characteristic D-banding patterns with a typical length
in the range of 64e67 nm (as shown by arrows in Fig. 1B inset),
similar to those of native collagen brils. Measurement of collagen
gel-coated PCL/Coll brous matrices by AFM further conrmed that

ultrane bers of collagen gel indeed lled the interstitial space of

PCL/Coll nanobrous matrices and led to a rather smooth surface
(Fig. 1D). In order to better characterize the roughness of these
matrices, roughness parameters (Rrms, root mean square roughness) were determined by using Nanorule software supplied with
AFM. As shown in Table 1, Fig. 1E and F, Rrms of PCL/Coll nanobrous
matrices alone was 596
15 nm, while it decreased to 269
7 nm
after coating with collagen gel. The actual difference on the surface
roughness between collagen gel-coated and noncoated PCL/Coll
nanobrous matrices could be even more pronounced without
artifacts that appeared in the images of PCL/Coll nanobrous
matrices (arrows in Fig. 1C).
3.2. Accelerated keratinocyte migration on collagen gel-coated
nanobrous matrices
In order to evaluate the effect of various nanobrous matrices on
keratinocyte migration, an in vitro wound-healing assay was performed by culturing the cells on various matrices with a wound
gap created in the middle (Fig. 2). After 24 and 48 h, staining of the
culture with methylene blue revealed that keratinocyte migratory
behavior was greatly correlated with their underlying matrices. As
shown in Fig. 2A and B, keratinocytes barely migrated into the
wound gap on PCL/Coll nanobrous matrices even after 48-h
culture. In contrast, keratinocyte migration was signicantly
enhanced on the surface of collagen gel-coated PCL/Coll nanobrous matrices, in which the wound gap was completely closed
by 48 h (Fig. 2A and B). In order to further elaborate whether the
accelerated motility of keratinocytes on collagen gel coated nanobers was due to chemical variation or the topographical changes,
wound healing assay was also similarly done on electrospun
collagen nanobrous matrices with an average diameter of
363
87 nm, PCL/Coll nanobrous matrices with adsorbed nonbrous collagen and glass cover slips (see Supplement Fig. 1).
Interestingly, neither pure collagen nanobrous matrices nor
collagen-adsorbed PCL/Coll nanobrous matrices promoted keratinocyte migration. However, high motility of keratinocytes was
observed on glass cover slips, similar to that on the collagen gelcoated nanobrous matrices.
3.3. Enhanced keratinocyte proliferation on collagen gel-coated
nanobrous matrices
Substrates that favor continuous cell proliferation are considered advantageous especially for re-epithelialization. Surface
chemistry of substrates has been well recognized for its crucial
contribution to the cell/substrate interactions [25,33e35]. However, it remains unclear whether surface topography of nanobrous
matrices also inuences keratinocyte adhesion and proliferation. In
this study, the proliferation of keratinocytes on both matrices was
determined by DNA assay for up to 7 days. Continuous cell proliferation was observed on both matrices and keratinocytes proliferated slightly faster on collagen gel-coated matrices compared to the
noncoated matrices on day 3 and 7 (Fig. 3).
3.4. Promotion of keratinocyte polarization by collagen gel-coated
nanobrous matrices
Keratinocytes change their morphology dramatically as they go
from stationary to migratory [17,36]. To determine the cell
morphology on nanobrous matrices with or without collagen gel
coating, keratinocytes cultured on these matrices for 24 h were
immunostained for the cytoskeleton protein, F-actin, and the focal
adhesion protein, vinculin. F-actin staining showed that keratinocytes exhibited a typical cobblestone-like morphology on the

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Fig. 1. Characterization of PCL/collagen nanobrous matrix alone (A, C, E) and with collagen gel coating (B, D, F). AeB) SEM micrographs; CeD) Two-dimensional AFM micrographs;
EeF) Three-dimensional AFM micrographs. Arrowheads: collagen gel ultrane bers; Asterisks: Electrospun PCL/collagen nanobers, Arrows in B: D-banding patterns of collagen
gel ultrane bers. Scale: 1 mm.

noncoated nanobrous matrices as characterized by the lack of

lamellipodia/lopodia and the existence of prominent cortical Factin after 2-day culture (Fig. 4A and C). Interestingly, most keratinocytes on the collagen gel-coated matrices became elongated
with at least one lamellipodium/lopodium presented (Fig. 4B and
D, arrowhead), consistent with the increased propensity for the
motile behavior. Some keratinocytes had more than one lamellipodium/lopodium accompanied by a more elongated and spindlelike morphology (Fig. 4D). The presence of more than one lamellipodium/lopodium is often related to their frequent switches of

Table 1
Characterization of PCL/collagen nanobrous matrices alone or with collagen gel
coating.
Composition

Average ber diameter (nm)

Roughness Rrms (nm)
a

Nanober

Gel coating

PCL/collagen
nanobers

PCL/collagen nanobers
coated with 3 mg/ml
collagen gel

331
112a
596
15a

55
26a
269
7a

Statistically signicant, p < 0.001.

the motion direction [37]. Staining of vinculin indicated that

various matrices indeed affected the spatial distribution of focal
adhesion plaques on the cell membranes. On the PCL/Coll nanobrous matrices, vinculin clusters mainly located on the circumferential region of the cell membrane along the nanobers (arrows
in Fig. 4E and G). On the collagen gel-coated nanobrous matrices,
vinculin clusters in keratinocytes were scattered in lamellipodia
(arrows in Fig. 4F and H) and stained vinculin clusters were slightly
smaller than those on the PCL/Coll nanobrous matrices (Fig. 4G vs.
H). To better quantify the morphology difference of keratinocytes
on various matrices, polarized keratinocytes from each group were
counted. It was found that more than 60% of keratinocytes on
collagen gel-coated nanobrous matrices remained polarized while
only about 20% of cells polarized on PCL/Coll nanobrous matrices
(Fig. 4I).

3.5. Upregulation of MMP-2 and MMP-9 activity in keratinocytes

by collagen gel-coated nanobrous matrices
Cell motility requires the release of cells from adhesion sites,
which is regulated, at least in part, by proteolysis of ECM molecules

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X. Fu et al. / Biomaterials 35 (2014) 1496e1506

Fig. 2. Keratinocyte migration on various nanobrous matrices. A) Cells were seeded on PCL/collagen nanobers without or with collagen gel coating with an insert in the middle.
After 12 h, the insert was removed to generate a 900 mm wound gap. Cells were allowed to migrate into the wound gap, and visualized after 24 and 48 h using methylene blue
staining. In order to determine the involvement of integrin b1 in regulating keratinocyte migration, anti-integrin b1 antibody was added to the culture to neutralize the integrin b1
function. Scale bar: 500 mm; B) Quantication of the wound gap distance between the front lines of the majority of migrating keratinocytes (n 4). **Statistically signicant,
p < 0.001.

Fig. 3. Keratinocyte proliferation plated on PCL/collagen nanobrous matrices alone or

with collagen gel coating for up to 7 days (n 4) *Statistically signicant, p < 0.05.

with MMPs [38]. Thus, the activities of MMP-2 and -9 released to

the supernatants of the culture were determined by gelatin
zymography for up to 7 days. As shown in Fig. 5A, the active form of
MMP-2 (i.e., 62 KDa) was detected at all three time points (day 1, 3
and 7) from the culture on collagen gel-coated nanobrous
matrices and reached its maximal activity on day 7. In contrast, the
supernatant from the culture on PCL/Coll nanobrous matrices
alone did not show detectable active MMP-2 at all the investigated
time points. With regard to MMP-9 activity, the active form, i.e.,
82 KDa was detected in all supernatant samples from both matrices
and its activity increased steadily over the extended culture
(Fig. 5A). However, a notably higher level of active MMP-9 was
detected in the cultures on collagen gel-coated matrices (Fig. 5A).
The inactive zymogen of either pro-MMP-2 or pro-MMP-9
remained constant in the supernatants for all the cultures during
the entire investigated times and showed no difference between
collagen gel-coated and noncoated nanobrous matrices. Semiquantitative analysis of the optical density of the bands from

X. Fu et al. / Biomaterials 35 (2014) 1496e1506

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Fig. 4. Morphology of keratinocytes on PCL/collagen nanobrous matrices alone or with collagen gel coating. A-H) Immunouorescent staining of intracellular cytoskeleton protein
of F-actin (Red) and nuclei with DAPI (purple/blue) (A, C: PCL/collagen nanober alone; B, D: with collagen gel coating), and focal adhesion proteins of vinculin (Green) (E, G: PCL/
collagen nanober alone; F, H: with collagen gel coating). Arrowhead: keratinocyte lamellipodium; arrow: spatial distribution of vinculin. I) Percentage of polarized keratinocytes
(n 3) **Statistically signicant, p < 0.001. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

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X. Fu et al. / Biomaterials 35 (2014) 1496e1506

Fig. 5. Gelatin zymograms showing gelatinase activities of keratinocytes on PCL/collagen nanobrous matrices alone or with collagen gel coating for up to 7 days. A) Representative
zymogram image; B) Quantication of MMP-2 and MMP-9 expression (n 3). *Statistically signicant, p < 0.05; **Statistically signicant, p < 0.001.

three separate experiments conrmed the consistency of the observations (Fig. 5B). These results suggest that collagen gel coating
indeed induces the activation of both MMP-2 and MMP-9.

synthesis in keratinocytes [40]. Our new results demonstrate that

collagen gel-coated scaffolds also enhance laminin-332 deposition,
which may facilitate keratinocyte migration.

3.6. Increase of laminin-332 (formerly termed laminin-5)

deposition by collagen gel-coated nanobrous matrices

3.7. Activation of integrin b1 in keratinocytes by collagen gelcoated nanobrous matrices

Laminin-332, a crucial ECM component secreted by keratinocytes is responsible for the repair of the damaged basement
membrane in skin wounds. It is deposited over collagen brils and
required for keratinocyte anchorage to and migration on the provisional basement membrane [36,39]. To determine whether
collagen gel-coated matrices may favor the deposition of endogenous laminin-332, immunostaining for laminin-332 was performed
on the samples cultured overnight. Compared to the limited
deposition of laminin-332 on non-coated matrices, a substantial
amount of laminin-322 deposited around and beyond the keratinocytes (strong uorescence was seen far from nuclei) on the
collagen gel-coated matrices (Fig. 6). Growth factors, cytokines and
lysophospholipids have been shown to stimulate laminin-332

Integrin b1 is one of the major ECM receptors present in keratinocytes. It is activated through outside-in and inside-out
signaling, and mediates keratinocyte adhesion and migration during the re-epithelialization [15,17,41e43]. To examine how various
nanobrous matrices regulate integrin b1 activation, immunouorescent staining was performed on the cultured keratinocytes to
detect the active conformation of integrin b1 using a monoclonal
antibody, HUST-4. A large amount of positive staining, i.e., active
integrin b1, appeared on the peripheral region of those keratinocytes cultured on collagen gel-coated nanobrous matrices
(Fig. 7A). In contrast, only a limited number of active integrin b1
plaques were found in the keratinocytes cultured on PCL/Coll
nanobrous matrices (Fig. 7A arrows). Noticeably, the size and

X. Fu et al. / Biomaterials 35 (2014) 1496e1506

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Fig. 6. Deposition of laminin-332 (laminin-5) by keratinocytes on PCL/collagen nanobrous matrices alone or with collagen gel coating. Scale bar: 100 mm.

distribution of active integrin b1 on keratinocyte membranes varied and depended on underlying matrix. On collagen gel-coated
matrices, elongated active integrin b1 evenly distributed on the
circumferential region of cell membrane, while round dots along
the ber orientation were observed on PCL/Coll nanobrous
matrices. Notably, some intracellular staining was also observed,
which required further studies in the future. Interestingly, the
expression and arrangement of active integrin b1 in those keratinocytes cultured on glass coverslip surfaces was similar to that on
the collagen gel-coated matrices (data not shown). Quantication
of the active integrin b1 by measuring the total uorescence intensity of the lysate of stained cells via multi-mode microplate
reader showed about two times the active integrin b1 on the
collagen gel-coated matrices in comparison to that on the noncoated ones (P < 0.01) (Fig. 7B). To further ascertain that collagen
gel-coated matrices indeed affect the activity of integrin b1, we
performed ow cytometry analysis. As shown in Fig. 7C, the level of
HUTS-4 binding (FITC staining) was found much higher on collagen
gel-coated matrices than that on the non-coated ones, consistent
with the immunostaining results.
3.8. Role of integrin b1 in keratinocyte migration on nanobrous
matrices
To determine the regulatory involvement of integrin b1 in keratinocyte motility on collagen gel-coated matrices, a woundhealing assay was performed on the keratinocytes with integrin
b1 activity neutralized by the neutralizing antibody. Apparently,
antibody neutralization signicantly inhibited keratinocyte
migration on the collagen gel-coated matrices. Even after 48 h, the
wound gap remained unclosed (Fig. 2A). This result was consistently observed at various seeding densities. This result together
with those of Fig. 7 indicated that integrin b1, whose activation was
stimulated by collagen gel coating, played a key role in regulating
keratinocyte migration on collagen gel-coated matrices.
3.9. Activation of Rac1 and Cdc42 in keratinocytes enhanced by
collagen gel-coated nanobrous matrices
Rac1 and Cdc42 of the Rho GTPase family are key downstream
signal-transduction molecules of growth factor receptors and
integrins. Their activation induces the formation of lamellipodia
and lopodia, thereby mediating integrin-dependent cell motility
[44]. Therefore, the activation of Rac1 and Cdc42 in cultured keratinocytes on both matrices was studied by afnity pull-down
assay using PBD- and GBD-GST fusion proteins. It was found that
the activation of Rac1 is markedly increased in keratinocytes
cultured on collagen gel-coated matrices, while that of Cdc42 is
slightly increased (Fig. 8). In contrast, there is no signicant change
of total Rac1 and Cdc42 on both collagen gel-coated and non-

coated matrices (Fig. 8). Thus, our results suggest that the
collagen gel-coated matrices stimulate keratinocyte motility likely
by activating integrin b1 and its downstream effectors Rac1 and
Cdc42.
4. Discussion
With regard to limited studies on the effect of nanobrous
matrices on keratinocytes, the present study was aimed to determine what kind of topographical features of nanobrous matrices
favor keratinocyte migration and what the associated underneath
mechanism is. Cultures of human keratinocytes on electrospun
PCL/Coll nanobrous matrices with or without type I collagen gel
coating showed that collagen gel coating, which bridged the
interstitial space of electrospun PCL/Coll nanobers with ultrane
bers, favored both cell proliferation and migration, important to
re-epithelialization. Further studies showed that collagen gel
coating increased active MMP-2 and -9 levels (consistent with the
increased MMP levels at early wound healing stages for cell
migration [16,45e47]), promoted endogenous laminin-332 deposition, activated integrin b1 and its downstream signaling molecules, Rac1 and Cdc42. This nding not only elaborates the possible
mechanism for enhanced keratinocyte motility on collagen-gel
coated PCL/Coll nanobrous matrices, but also indicates the similarity for keratinocytes on collagen gel-coated nanobers and
wound provisional matrix. Thus, electrospun nanobrous matrices
coated with collagen gel may also be used as an in vitro model for
provisional matrix to study the cellematrix interactions relevant to
wound healing process.
Culturing keratinocytes on various collagen-containing nanobrous matrices showed that only collagen gel-coated PCL/Coll
nanobrous matrices elevated keratinocyte motility, while neither
pure electrospun collagen nanobrous matrices nor electrospun
PCL/Coll nanobrous matrices adsorbed with nonbrous collagen
showed such stimulatory effect (Supplement Fig. 1), indicating the
surface topography of matrices indeed play an essential role in
regulating the migratory activity of keratinocytes. Different from
the large pores (w2 mm) in PCL/Coll electrospun nanober
matrices, collagen gel coating on the surface of PCL/Coll nanobers
led to the formation of a secondary bril network composed of
ultrane bers (55
26 nm in average diameter) with interstitial
pores less than 200 nm. This ultrane bril network turned the
surface of PCL/Coll nanobrous matrices into a relatively smooth
one (Fig. 1B and D). Keratinocytes are relatively smaller with short
lamellipodia in comparison to dermal broblasts. On PCL/Coll
electrospun nanobrous matrices, the interber distance may be
too large for keratinocytes to form sufcient cell/matrix contacts to
migrate; as a result, fewer focal adhesion plaques would be
developed. On the other hand, collagen gel-coated matrices provide
a relatively smooth surface for keratinocytes to adhere, which may

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X. Fu et al. / Biomaterials 35 (2014) 1496e1506

Fig. 7. Expression of active b1 integrins in keratinocytes on PCL/collagen nanobrous matrices alone or with collagen gel coating. A) Immunouorescent staining of active integrin
b1. B) Quantication of the lysates of cells uorescently stained for active b1 integrins with a uorometer (n 3). **Statistically signicant, p < 0.001. C) HUTS-4 binding determined
via ow cytometry (n 3).

allow the formation of more contact points. Indeed, immunostaining for vinculin did show fewer focal adhesion points formed
on keratinocytes cultured on non-coated matrices and their distribution pattern closely correlated with the spatial arrangement of
electrospun bers (Fig. 4E and G). In contrast, more uorescently
stained vinculin was observed in the keratinocytes cultured on
collagen gel-coated matrices (Fig. 4F) and its distribution followed a
radial pattern for individual cells (Fig. 4H).
As a membrane cytoskeletal protein, vinculin links intracellular
actin laments to the integrin components of focal adhesion
complexes [48], thus its distribution would greatly affect cell
morphology. Staining for F-actin revealed distinct cell morphology
between the coated and non-coated matrices. Keratinocytes on the
collagen gel-coated matrices showed more polarization and the
formation of lamellipodia (Fig. 4A vs. B, and Fig. 4I), indicating the
possible presence of more migrating keratinocytes [36]. We also
found that gel-coated matrices signicantly increased the active
forms of MMP-2 and -9 (Fig. 5), which are known for their participation in epithelial cell migration by cleaving ECM molecules in
concert with other MMPs like MMP-1 [16,45]. MMP-2 was reported
to localize in the cell/ECM contactlike structures of migrating

keratinocytes [47] and modify ECM molecules to induce cell

migration. It has also been reported that MMP-2 colocalizes with
laminin-332 by binding to its g2 chain [49] and subsequently
cleaves the g2 subunit at residue 587, exposing a putative cryptic
promigratory site on laminin-332 that triggers cell motility [50]. In
this study, a substantial amount of laminin-332 was indeed
detected in the culture on gel-coated PCL/Coll nanobrous matrices
while little was seen on non-coated matrices (Fig. 6).
Deposition of laminin-332 onto gel-coated PCL/Coll nanobrous
matrices would switch the adhesion and signaling of keratinocytes
from collagen-dependent to laminin-332 dependent, mediated by
different integrins [51]. Keratinocytes then use these freshly
deposited laminin-332 in locomotion. Since integrin b1 is a known
receptor for this ligand, it is reasonable to assume that ECMinduced integrin b1 expressions can differentially regulate keratinocyte motility. It was found that keratinocytes cultured on
collagen gel-coated matrices showed a signicant increase of active
integrin b1 and the neutralization of its activity using blocking
antibody partially abolished the facilitated migration (Figs. 2 and 7),
consistent with prior mouse genetic studies indicating the participation of b1 integrins in epidermal growth and repair [41]. A

X. Fu et al. / Biomaterials 35 (2014) 1496e1506

1505

other hand, modication of such nanobrous matrices with a secondary ultrane collagen brous network with the ber diameter
down to 55
26 nm can signicantly stimulate keratinocyte
migration. Our further studies have shown that such a secondary
brous network stimulates the adhesion, proliferation and migration of keratinocytes by regulating the activation and distribution
of integrin b1 and the downstream effectors of Rac1 and Cdc42,
facilitating the deposition of laminin-332, and promoting the
expression of active MMPs (MMP-2 and 9). Clearly, nanoberinduced distinct keratinocyte responses are closely regulated by
the topography of the nanobrous matrices, which might be as
important as the chemistry of the matrices.
Author disclosure statement
No competing nancial interest.
Acknowledgments
This investigation was sponsored by the Grant Number 1R21
AR056416 from NIAMS. Dr. Xiaoling Fu was supported by the
Innovation & Entrepreneurship Doctoral Fellowship from the Stevens Institute of Technology.
Appendix A. Supplementary data
Supplementary data related to this article can be found online at
http://dx.doi.org/10.1016/j.biomaterials.2013.11.013.

Fig. 8. Activation of Rac1 and Cdc42 in keratinocytes enhanced by collagen gel-coated

PCL/collagen nanobers matrices. A) Activation of Cdc42 and Rac1 in keratinocytes
cultured on PCL/collagen nanobrous matrices alone or with collagen gel coating were
determined by afnity pull-down assay. The total Cdc42 and Rac1 were measured by
western blot analysis. Actin was used as a loading control. (B) Protein bands were
quantied by densitometry and plotted.

Possible explanation to b1 integrin-induced cell motility is that b1

integrin triggers a more motile phenotype via activating small
GTPases Rac1 mediated by FAK/Cas/Crk signaling pathway and
Cdc42. It is known that Rac1 and Cdc42 control cell migration
through increasing the formation of motile actin-based structures
including lamellipodia and helping establish cellular polarity in
response to ECM [43,52,53]. We indeed observed higher activation
of Rac1 and Cdc42 in keratinocyte on collagen gel-coated matrices
(Fig. 8). Clearly, b1 integrins act as a critical mediator for outside-in
signal transduction for keratinocytes specically on the collagen
gel-coated matrices.
Our studies highlighted the important role of topographical
feature of nanobrous matrices in regulating keratinocytes for their
re-epithelialization function conveyed by integrin b1 signal transduction, which is mediated via Rac1 and Cdc42. Clearly, the ndings
would provide an insightful understanding of the regulatory
mechanisms of nanober-modulated phenotypic expression of
human keratinocytes for appropriate wound repair, but also offer
an interesting paradigm to study the potential contributions of
various ECM-like brous matrices to in vivo wound healing
processes.
5. Conclusion
In this study, we explored keratinocyte motility and other behaviors related to wound healing on various nanobrous matrices.
Apparently, collagen-containing nanobers with diameters of
331
112 nm do not support human keratinocyte migration. On the

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