Cell Migration and Proliferation During the In

Vitro Wound Repair of the Respiratory

Epithelium
Jean-Marie Zahm,* Herve Kaplan, Anne-Laure He rard, Fabrice Doriot,
Denis Pierrot, Pascal Somelette, and Edith Puchelle
IN SERM U 314,IFR53,C H U M aison Blanche,Reim s,France
The respiratory epithelium is frequently injured by inhaled toxic agents or by
micro-organisms. The epithelial wound repair represents a crucial process by
which surface respiratory cells maintain the epithelial barrier integrity. The repair
process involves both cell migration and proliferation, but as yet, the kinetic of
these two mechanisms has not been extensively studied. Using an in vitro model of
human respiratory epithelium wound repair, proliferative cell immunouorescent
staining and a computer-assisted technique allowing the tracking of living cells, we
studied the cell proliferation and migration during the wound repair process.
Respiratory epithelial cells were dissociated from human nasal polyps and cultured
on a collagen I matrix. At conuency, a chemical wound was made on the culture.
We observed that the cell mitotic activity peaked at 48 h after wounding (23% of
the cells) and mainly concerned the cells located 160 to 400 m from the wound
edge. The migration speed was highest (35 to 45 m/h) for the spreading cells at the
wound edge and progressively decreased for the cells more and more distant from
the wound edge. The temporal analysis of the cell migration speed during the
wound repair showed that it was almost constant during the rst 3 days of the repair
mechanism and thereafter dropped down until the wound closure was completed
(after 4 days). We also observed that over a 1-hour period, the intra-individual and
interindividual variation of the cell migration speed was 43% and 37%, respec-
tively. These results demonstrate that cell proliferation and cell migration during
respiratory epithelial wound repair are differently expressed with regard to the
cell location within the repairing area. Cell Motil. Cytoskeleton 37:3343,
1997.
r
1997 Wiley-Liss, Inc.
Key words: airway wound; migrating cells; proliferative cells; spreading cells; imaging analysis; cell
tracking
INTRODUCTION
The respiratory epithelium is frequently injured by
inhaled toxic agents or micro-organisms, leading to the
alteration of the epithelium barrier integrity. Whatever the
source of injury, the response of the respiratory surface
epithelium to an acute injury can be characterized by a
succession of cellular events varying from the loss of
surface epithelial impermeability to the desquamation of
cells from the epithelium. The epithelial wound repair
represents a crucial process by which the remaining
surface respiratory cells restore the epithelial barrier
integrity. Numerous models have been used to explore the
capacity of the respiratory epithelium to repair following
injury. Most of these models have been developed in vivo
in different animal species and have allowed the deduc-
tion that denudation of the airway epithelial surface leads
Contract grant sponsors: Association Francaise de Lutte Contre la
Mucoviscidose (AFLM), Pole Technologique Regional GBM, IN-
SERM(Contrat Normalise dEtude Pilote), Ministe`re de lEnseignement
Superieur et de la Recherche.
*Correspondence to: Jean-Marie Zahm, INSERM U314, CHU Maison
Blanche, 45, rue Cognacq-Jay, 51092 Reims Cedex, France.
Received 19 July 1996; accepted 18 November 1996.
Cell Motility and the Cytoskeleton 37:3343 (1997)
r
1997 Wiley-Liss, Inc.
to the surrounding epithelial cell migration and prolifera-
tion [Gordon and Lane, 1976; Keenan et al., 1982;
McDowell et al., 1979; Nikula et al., 1988; Shimizu et al.,
1994]. However, the kinetic of these two major processes
during the wound repair has not been extensively studied,
due to the difficulty introduced by in vivo models. In
addition, in all these animal models of injury and repair,
the inammatory stimulus engages cellular factors and
serum-derived molecules into the process of repair.
To overcome the complex association of factors in
the in vivo models, culture models of respiratory epithe-
lial cells allow more clearly the identication of the
cellular and molecular alterations associated with the
wound repair processes [Zahm et al., 1991, 1993]. At the
molecular level, alterations in cytoskeletal protein pat-
terns, binding of growth factors and interactions with
extracellular matrix proteins and metalloproteinases
through specic cells receptors (integrins), can govern
the cell migration in the repairing area. The attened cells
which migrate into the wounded site change their pheno-
type, becoming poorly differentiated cells expressing
keratin 14 and vimentin intermediate laments [Shimizu
et al., 1994; Buisson et al., 1996a]. The cell spreading and
migration which occur during wound repair depend on
cell-matrix interactions mediated by bronectin and one
of its cellular receptors, the a5b1 integrin [Herard et al.,
1996]. Recent results have also documented the increase
and localization of gelatinolytic metalloproteinases asso-
ciated with the migratory process of epithelial cells
during the respiratory epithelium repair [Buisson et al.,
1996b]. However, up to now, the specic role of cell
migration and cell proliferation in the wound repair
process of the respiratory epithelium has not been exten-
sively documented.
In the present study, we used an in vitro wounding
model of dissociated respiratory epithelial cells cultured
from human nasal polyps to evaluate the dynamics of the
cell migration and proliferation during wound repair. We
also reported in the present work, a novel method, using
image analysis, to provide data on the migration kinetics
of either individual cells or a cell population during the
wound repair process. We observed that the cell mitotic
activity peaked at 48 h after wounding. The temporal
analysis of the cell migration speed showed that, despite
large intra-individual and interindividual variations, the
overall migration speed of the cell population at the
wound edge remained fairly constant during the rst 3
days of repair and decreased progressively up to the
wound closure.
MATERIALS AND METHODS
Respiratory Epithelial Cell Culture
Nasal polyps were obtained from patients undergo-
ing nasal polypectomy and were immediately immersed
in RPMI 1640 culture medium (Gibco, Grand Island,
NY). The nasal tissue was then dissociated by enzymatic
digestion (pronase 0.1%) for 12 h. The dissociated
surface epithelium was removed from the tissue by gentle
agitation and enzymatic digestion was stopped by adding
10% calf serum (Seromed, Biochrom, Berlin, Germany).
The cellular pellet collected after centrifugation at 150 3
g for 10 min was constituted by isolated mucous cells,
ciliated or basal cells, small clumps of reaggregated cells
and small epithelial sheets. These cells were resuspended
in a serum-free medium and were plated on a type I
collagen gel matrix prepared from rat tail tendons accord-
ing to the technique developed in our laboratory and
earlier described by Chevillard et al. [1993]. The cells
were cultured in an humidied atmosphere containing
95% air and 5% CO
2
in RPMI 1640 medium supple-
mented with 1 g/ml insulin (Sigma, LIsle DAbeau,
France), 1 g/ml transferrin (Sigma), 10 ng/ml epidermal
growth factor (Serva, Heidelberg, Germany), 0.5 g/ml
hydrocortisone (Sigma), 10 ng/ml retinoic acid (Sigma),
100 U/ml penicillin, and 100 g/ml streptomycin. The use
of type I collagen gel and growth factor-supplemented
culture medium is the most suitable condition to achieve
differentiated cultures of human nasal epithelial cells
[Chevillard et al., 1993].
In Vitro Epithelial Wounding
After 3 days in culture, when the cells had reached
conuency, the culture medium was removed from the
culture dish. A2-l drop of NaOH 0.1 M was deposited in
the center of the culture dish and immediately diluted in 1
ml of culture medium. The cells in contact with the NaOH
drop desquamated from the collagen I matrix, creating a
circular wound of about 30 mm
2
in area. The wounded
culture was then rinsed with 1 ml of culture medium and
incubated in air with 5% CO
2
at 37C.
Wound Area Determination
Every 12 h during the wound repair process, the
wounded culture dish was placed on the stage of an
inverted microscope (Nikon TMS-F) equipped with a 31
objective and a video CCD camera (Cohu 4700). The
video image of the wound was displayed on a video
monitor and the wound edge was drawn manually. From
this drawing, the wound area was determined and ex-
pressed as a function of time.
Cell Proliferation
For cell proliferation quantitation, wounded cul-
tures were cryoxed every 24 h throughout the wound
repair process. The central area of the culture (about 200
mm
2
in area) in which the wound was performed, was cut
34 Zahm et al.
out from the culture dish, embedded in OCT compound,
immersed in liquid nitrogen for 5 min and then kept at
280C. Thick sections (5 m) perpendicular to the
wounded culture were obtained using a Reichert Cryocut,
sliced on a glass slide and dehydrated in air. For
immunostaining, the sections were immersed in methanol
at 220C for 5 min and rinsed in 0.1 M phosphate-
buffered saline (PBS). Each section was then incubated
for 1 h with a mouse monoclonal antibody (MIB-1,
Immunotech, France) which recognizes the Ki67 nuclear
antigen and which was diluted 1/50 in 0.1 M PBS and
thereafter incubated with a biotin-conjugate goat anti-
mouse IgM, diluted 1:25 in 0.1 M PBS for 1 h. The cells
were then stained with streptavidin-FITC for 30 min. The
sections were mounted in glycerol-PBS-1,4 diazabicyclo
2-2 octane and observed under a Zeiss Axiophot micro-
scope equipped with epiuorescence illumination. Succes-
sive microscopic elds of 40 m in length radiating out
from the wound edge were observed. In each eld, the
number of cells being positively stained for the Ki67 was
quantied and expressed as a percentage of proliferative
cells with regard to the total number of cells.
Video Recording of the Cell Migration
Immediately after wounding, cell cultures were
incubated for 15 min with Hoechst 33258 (Sigma) at 0.1
mg/ml in culture medium, allowing the uorescent stain-
ing of the nucleus in living cells. The culture was then
washed twice with culture medium to remove the excess
of uorescent dye. Following that, the wounded culture
dish was placed on the stage of a Zeiss IM35 inverted
microscope and was enclosed in a small transparent
culture chamber (Climabox) with 5% CO
2
in air at 37C.
The microscope was equipped with an epiuorescence
illumination (Hg lamp) through an excitation lter at 360
nm, and an emission lter at 510 nm, and with a low level
SIT camera (Lhesa 4036). A special electronically oper-
ated shutter in the excitation light path was developed in
order to automatically illuminate the culture at short
periods of time, and to simultaneously record the uores-
cent images. This device, based on timing semiconductor
circuits, allowed the denition of the recording time of
each video sequence (from 1 to 99 s) and the time interval
between two successive sequences (from 1 to 99 min).
Experimental Procedure
Video recordings of the uorescent-stained cell
nucleus were performed following three sets of experi-
ments during the wound repair process. In the rst set of
experiments, we recorded for 2 s every 12 h the cell
nucleus at the wound edge, throughout the wound repair
process. In the second set of experiments, at day 2 of the
wound repair, we recorded successive microscopic elds
which were radiating out from the wound edge. In the
third set of experiments, at day 2 of the wound repair, we
recorded for 2 s every 5 min, the uorescent nucleus of
cells in the same microscopic eld at the wound edge.
From these video recordings, we used an image analysis
technique that we developed to analyze the migration of
individual cells forming continuous sheets. In one experi-
ment, using phase-contrast illumination, we continuously
recorded the cells at the wound edge for a 10-min period
in order to visualize the cell spreading.
Cell Tracking and Cell Migration Measurement
The images were digitized as a 766 3 574 pixels
and 24-bit array from the video recordings, using a
Sparc-Classic workstation equipped with a XVideo card
(Parallax Graphics, Santa Clara, CA). Before treatment,
the images were reduced to 512 3 512 pixels and 8-bit
grey levels. We developed a software in C language using
the Xlibrary (version 1.1 of the XWindow system) and a
tool kit available in XView (Sun Microsystems, Moun-
tain View, CA). The software is user-interfaced and has
three main functions: (1) the detection of the cell nucleus,
(2) the computation of the trajectories of the nucleus, and
(3) the analysis of the nucleus trajectories.
The detection of the cell nucleus can be done
manually by using the computer mouse or automatically
by using image segmentation methods based on threshold-
ing. The tracking of several nuclei together is done by
using trajectory-based methods [Aggarwal and Nandhaku-
mar, 1988]. The general scheme of these algorithms is to
minimize the mean cost of all the computed trajectories,
using one or several parameters such as the regularity of
cell motion, the low deviation of the cell trajectory or the
nearness of the successive positions of the cells. For the
main part, we used nearness and low deviation of the
trajectories as parameters of minimization for several
successive images and nearness parameters for only two
successive images. After trajectory computation, the
software draws all the trajectories of the selected nucleus.
From this drawing, each nucleus trajectory can be se-
lected individually and the migration speed is calculated
from one nucleus position to the other. The mean
migration speed for all the nucleus trajectories is also
calculated. The software is able to track a cell population
of about 100 cells together. The computation time for 100
cells in eight images is about 2 s using an Unix
Workstation (Sun Microsystems).
Statistical Analysis
All the data are expressed as mean 6S.D. (standard
deviation of the mean). The unpaired Students t test was
used to determine signicance (dened as p , 0.05).
Curve tting was performed on a Macintosh Quadra 650
using CA-Cricket Graph III (Computer Associates, New
York, NY).
Cell Proliferation and Migration in Wound Repair 35
RESULTS
Wound Repair Process
Human surface respiratory epithelial cells cultured
on a type I collagen reached conuency within 2 to 3
days. The contact of a drop of NaOH with a conuent
respiratory epithelial cell culture induced a localized
injury characterized by the cell lysis and desquamation
and the denudation of the collagen matrix on which the
cells were growing. The chemically induced wounds in
the respiratory epithelial cell culture consisted of circular
holes. Microphotographs taken using an inverted micro-
scope at low magnication (31 objective), immediately
after wounding and thereafter every 30 h during wound
repair, show the progressive closure of the wounded
surface (Fig. 1). The early reepithelialization pattern
displayed by the respiratory cells immediately after
injury, appeared to involve the spreading of the cells at
the wound edge and the movement of cell sheets toward
the wound [Zahm et al., 1991]. The migration process is
uniformly distributed around the wound surface as sug-
gested by the concentricity of the successive wound
surfaces. A regular feature which progressively appeared
during the wound repair is the puckering of cell sheets in
the repaired area (Fig. 1, t90). The observation of
semithin sections of wounded cultures revealed that the
puckers radiating around the wound are made of multilay-
ered cells.
The mean wound surface plotted versus time de-
creased sharply up to the 50th hour of repair (Fig. 2). A
third degree polynomial t through the experimental data
gave the following curve equation:
Wa
(t)
5 27.99 2 0.37t 2 1.28 3 10
23
t
2
2 2.14 3 10
25
t
3
where Wa is the wound area and t the time of repair. As
shown by the equation of the t curve, the rst-order
coefficient corresponded to the linear part of the t curve.
The decrease in wound area appeared therefore linear up
to 50 h of repair due to the fact that the second- and
third-order coefficients of the t equation interfered only
about 10% in the rate of decrease. However, on and after
the 60th hour of repair, the third-order coefficient acts as a
factor attening out the curve and alters the rate of
decrease by 16%, 39% and 77% at 60, 80 and 100 h of
repair, respectively. This observation indicates that the
repair process becomes slower when most of the injured
surface was recovered by the repairing cells.
Cell Proliferation
In order to assess whether the in vitro model of
respiratory epithelium wound repair involved cell prolif-
eration, wounded cultures at different days of the repair
process were immunouorescently stained for the nuclear
antigen Ki67 which is a marker of cycling cells. The
micrograph in Figure 3a shows a number of Ki67-
positive cells in the culture 2 h after wounding (day 0). As
shown in Figure 3b, the number of proliferative cells in
the repairing area increased dramatically at 48 h after
wounding (day 2). When the wound closure was com-
pleted (day 4), the number of proliferative cells fell down
to a value similar to that obtained at day 0 (data not
Fig. 1. Set of low magnication (31 objective) micrographs showing
the in vitro wound repair process of the respiratory epithelium. The rst
micrograph (t0) was taken immediately after wounding. The following
micrographs were taken every 30 h during the wound repair. The
progressive closure of the wound is observed. Bar 51 mm.
Fig. 2. Temporal evolution of the wound surface during the wound
repair process of the respiratory epithelial cells, whose micrographs are
shown in Figure 1. Each point represents the mean 6 S.D. of three
experiments. The curve corresponds to a third-degree polynomial t
through the experimental data. The decrease in wound area (0.37 mm
2
per hour) appears linear up to 60 h and then rapidly attens out up to
wound closure.
36 Zahm et al.
shown). Figure 3c illustrates the variations in the prolifera-
tive activity of the cells in the repairing area. The
percentage of proliferative cells was 7.1 67.0% at day 0,
increased signicantly ( p , 0.05) up to 23.0 6 25.6% at
day 2 and decreased to 2.8 6 3.0% at day 4 when the
wound closure occurred.
In order to assess where the proliferative cells were
located, we quantied their number in areas more and
more distant from the wound edge. The variation at day 2
in the percentage of proliferative cells in relation to the
distance from the wound edge is represented in Figure 3d.
A slight increase in the number of proliferative cells was
observed at a short distance from the wound edge. The
cells located in the repaired area at a distance of 80 to 320
m from the wound edge were characterized by a
signicant increase ( p , 0.05) in their proliferative activ-
ity. The decrease in proliferation between 160 m and 240
m was not signicant compared to 160 m, but the
increase in proliferation at 320 m was signicantly
higher ( p , 0.05) as compared to the proliferation at 250
m. On the contrary, the proliferation of cells located at a
distance higher than 400 m from the wound edge
dramatically decreased to only 12%.
These results demonstrate that the proliferative
activity is maximal at day 2 of the repair process and
mainly concerns the cells located within an area behind
the rst cell rows of the wound border.
Cell Spreading
To analyze the dynamics of cell motility at the
wound edge of wounded respiratory epithelial cell cul-
tures, video recordings of the wounded cultures were
made under phase-contrast illumination. From 60 images
recorded every min for 1 h, a movie was produced,
allowing the visualization of lamellipodia dynamics of
the cells located at the wound edge. Four pictures
Fig. 3. Proliferative activity of respiratory epithelial cells during the in
vitro wound repair. The proliferative cells were detected by immunou-
orescence of the Ki67 antigen. a: A few Ki67-positive cells are
observed at day 0 of wound repair. b: Forty-eight hours after
wounding, numerous Ki67-positive cells are present in the repairing
area. c: Percentage of proliferative cells at day 0, day 2 and day 4 of the
wound repair process. A signicant increase in the percentage of
proliferative cells was observed at day 2 ( p , 0.05). At wound closure
(day 4), the percentage of proliferative cells was not signicantly
different as compared with day 0. Each bar represents the mean
percentage 6 S.D. of proliferative cells quantitated on at least 12
different repairing areas. d: Spatial evolution, at day 2, of the
percentage of proliferative cells in the repairing area. A signicant
increase ( p , 0.05) in the percentage of proliferative cells was
observed at a distance of 80 to 320 m from the wound edge. Each
value represents the mean 6S.D. of three measurements.
Cell Proliferation and Migration in Wound Repair 37
extracted from the movie every 3 min are presented in
Figure 4. In order to better visualize the front of the cell
lamellipodia, its border was underlined by a white
drawing. The lamellipodia formation was a characteristic
feature of the cells at the front row of the wound edge.
The lamellipodia remained strictly localized at the head
or front of migrating cells. We can easily observe that the
shape of the cell edge is continuously modied during the
spreading process, but the cell functional polarity (mo-
nopolar) remained constant throughout the repair process,
leading to the unidirectionality of cell movement.
Cell Migration
To track the migratory cells repairing the wound,
we stained the cell nuclei with Hoechst uorescent dye.
The uorescent images obtained under these conditions
are less complex than phase-contrast images (see Fig. 4)
and therefore are more easily analyzed with the image
analysis software specically developed for the cell
velocity measurement (Fig. 5). The micrographs in
Figure 5a show cell nuclei stained with the Hoechst dye at
the edge of a wound at day 2 of the wound repair. Figure
5b represents the same nuclei recorded 60 min later. The
trajectories determined from the eld analyzed every 15
min over the 60-min period of observation were fairly
straight (Fig. 5c and d). As shown in Figure 5d, the
software that we developed allowed us to calculate the
angular deviation (a1, . . . , a4) from the horizontal of the
successive trajectories of the nuclei. The mean deviation
angle for all the selected nuclei was 0.30 60.11 rd, which
conrmed the uniform directionality of the cell migra-
tion. These trajectories represent a typical behavior of the
radian migrating cells which actively participated to the
wound repair process.
Fig. 4. Series of phase-contrast images, taken at 3-min intervals, of respiratory epithelial cells migrating
and extending lamellipodia at the wound edge. The leading edge of the cell lamellipodia has been
visualized by a white line. The cells located at the wound edge are characterized by a functional polarity
(monopolar) with a continuously changing lamellipodia shape. Bar 510 m.
38 Zahm et al.
The migration speed of the cells at the wound edge
during the wound repair process is represented in Figure
5e. We measured an almost constant speed (35 to 40
m/h) for up to 60 h of repair, followed by a signicant
decrease from hour 72 until the wound closure. When
compared with the wound area decrease reported in
Figure 2, the time of 60 h may be signicant, because it
roughly corresponded to the time up to where the
decrease in wound area was roughly constant. Conse-
quently, the decrease in the cell migration velocity
induced an alteration in the wound area decrease rate.
Figure 5f represents the mean migration speed of a
cell population measured every 5 min for 1 h at the wound
edge at day 2. The high standard deviation of the
migration speed calculated every 5 min for the cell
population indicated that huge variations (interindividual
variation 5 37%) in cell migration speed were observed
for different cells during the same time period. In
Fig. 5. Quantitative evaluation of cell migration velocity. a: Fluores-
cent staining with a DNA uorescent dye (Hoechst 33258) of the
nucleus of respiratory epithelial cells at the edge of a wounded culture
at day 2 of the wound repair process. b: The same cells recorded 1 h
later. c: Trajectories of cell nuclei recorded over a 60-min period. d:
Detailed trajectories of a particular nucleus analyzed every 15 min for 1
h. The angle a represents the deviation with the horizontal of the
nucleus trajectory. e: Temporal evolution of the migration velocity of
cells at the wound edge throughout the wound repair process. A
signicant decrease in the migration speed was observed at 72 h. Each
bar represents the mean 6 S.D. of 15 measurements. f: Temporal
evolution for 1 h of the migration speed of cells at the wound edge. The
same 15 cells were recorded every 5 min for 1 h. Huge variations were
observed from one cell to the other. Each bar represents the mean 6
S.D. of the migration speed for the 15 cells.
Cell Proliferation and Migration in Wound Repair 39
addition, the mean coefficient of variation of the migra-
tion speed for the same cell measured every 5 min for a
1-h period was 43%(5intra-individual variation), indicat-
ing that the migration speed of one given cell was not
constant over a 1-h period.
The mean migration speed of cells measured in
successive areas progressively more distant from the
wound edge is represented in Figure 6. We observed that
the cell migration speed continuously and signicantly
( p , 0.05) decreased with the increase in the distance
from the wound edge. The epithelial cells at the wound
edge moved faster than the cells which were distant from
the wound edge. At a distance higher than 1.6 mm from
the wound edge, the cell migration was no longer
detectable.
DISCUSSION
The experiments described in the present work
show that the respiratory epithelium is able to repair
wounds through a combination of cell migration and cell
proliferation. Cell proliferation was quantied through
the Ki67 antigen which is a marker of cycling cells, i.e.,
cells in G1, S, G2 and M phases. In our in vitro model of
respiratory epithelial wound repair, cell proliferation took
place preferentially in the repairing area, with a mitotic
activity which peaked at 48 h. When the wound closure
was completed, the mitotic activity subsided. These
results are consistent with the recent results of Shimizu et
al. [1994] who demonstrated a similar pattern of mitotic
activity (24% of proliferative cells at 48 h after wound-
ing) in the in vivo wound repair process of the rat tracheal
epithelium.
The major event in the wound repair process is the
cell spreading and migration which allows the epithelium
recovery of the denuded collagen matrix. Time-lapse
video recordings associated with computer methods al-
low the continuous monitoring of the process of cell
spreading and migration.
As seen in Figure 4, under phase-contrast micros-
copy observation, it is difficult to individualize the cells
and to monitor their movement in a conuent cell culture.
To overcome this problem, we stained the cell nucleus
with a DNA dye (Hoechst 33258) which is excitable by
UV illumination. Taking into account what we have
previously demonstrated, that the duration of UV illumi-
nation can induce dramatic changes in the physiological
state of the cells [Zahm et al., 1994], we used an
electronic timer which allowed us to reduce the UV
illumination time to only a few seconds for each video
recording.
To track the moving cells, we used trajectory-based
methods [Aggarwal and Nandhakumar, 1988] which
commend several assumptions. Firstly, the nuclei, which
are used as markers of the cells, are considered as a rigid
body and are indistinguishable from each other. Secondly,
each nucleus is tracked using only one representative
point: the center of the nucleus. While the cells are
tracked using the center of the nucleus as a single marker,
only translation movements of the cells are observable.
Other movements, such as cell rotation, for example, are
not detectable. The trajectory-based method raises the
problem of the occlusions which concern the objects
visible in an image and no longer visible in the following
image of the sequence. We also have to consider the case
of objects which are not present in an image and become
visible in the following images. Some algorithms dealing
with the occlusions have been described [Fletcher et al.,
1991; Rangarajan and Shah, 1991; Sethi and Jain, 1987],
but these algorithms generally only take into account the
missing objects in successive image sequences. In the
images from our wound repair model, the number of
nucleus in an image is continuously increasing through-
out the set of images. To overcome this problem, we used
an algorithm which considered that the number of tracked
nuclei was constant in all of the successive images. With
that purpose, the nuclei were selected manually in the set
of images. This is done easily and quickly using the
computer mouse and is facilitated by the cell nucleus
staining which made the nuclei clearly visible in the
images.
Two sets of methods for establishing the correspon-
dence between the selected points are available. The rst
methods are based on the regularity of the migration of
Fig. 6. Spatial evolution of the migration speed of cells at day 2 of the
wound repair process. The cell migration speed progressively and
signicantly ( p , 0.05) decreased with the increase in the distance
from the wound edge. Each bar represents the mean 6 S.D. of 15
measurements.
40 Zahm et al.
the objects [Sethi and Jain, 1987] and are called smooth-
ing methods. To determine the trajectories of the nuclei,
we have to maximize the smoothest set of trajectories.
Smoothing can be controlled by several factors such as
nearness, speed of motion or deviation of the trajectories.
The second methods are predicting methods [Fletcher et
al., 1991], by which the estimation of the position of the
objects in the image is computed using information from
the partial trajectory of the objects. The object which is
nearest to the calculated position is chosen and the
predicting factors corrected according to the knowledge
of this new position. The process must be initiated using
the optical ow method [Uras et al., 1988] or a smoothing
method, on at least three images.
Several techniques have been described for analyz-
ing cell motility, but most of these techniques are applied
to isolated migrating cells. Tatsuka et al. [1989] described
a method for quantitative measurement of postconuent
cell populations, but the latter authors measured a motility
index of a cell population and not the locomotion speed of
individual cells. The main advantage of the computer-
associated technique that we have developed is that we
can track a population of conuent cells over a long
period of time, and measure their individual migration
speed, allowing the study of the factors controlling and
coordinating the locomotory machinery.
The ability of cells to move is crucial for many
biological processes, such as embryogenesis and wound
healing. In this current study, we analyzed the cell
migration during the wound repair process of the respira-
tory epithelium. The cells at the wound edge are character-
ized by a mean migration speed ranging from 35 to 45
m/h during the rst 60-h period of the wound repair
process. We have previously shown [Zahm et al., 1992]
that in a smaller mechanically made wound (0.03 mm
2
in
surface), the cell migration speed reached 26 m/h. But in
this latter wound model, the wound closure occurred
within 6 to 8 hours. The decrease in the migration speed
measured at the end of the wound repair process is most
likely due to contact inhibition effects. Recently, Lee et
al. [1994], by analyzing the measurements of cell path, in
conjunction with local cell velocity and the distance to the
nearest neighbor of a cell, have demonstrated, in detail,
the behavior of a cell as it migrates and interacts with
other cells. These authors showed that as two cells
approach each other, their speed decreases, whereas when
a cell speeds away from its neighbor, its speed increases
quickly. This behavior of neighboring cells could also
explain the huge variations in cell locomotion that we
observed when analyzing the local movement of a cell
population at the wound edge for a short period of time
(Fig. 5).
In analyzing the migration of cell populations
located at an increasing distance from the wound edge,
we observed a negative relationship between the cell
migration speed and the distance from the wound edge:
the cells located far from the wound edge moved more
slowly than the cells located at the wound edge. As shown
in Figure 3, the maximum increase in cell proliferation
occurred in the intermediate area of the repaired wound.
Lee et al. [1994] have shown that an increase in cell
density induced by cell proliferation is immediately
accompanied by the decrease in the average speed of
migration of a cell population. In addition, several years
ago, Abercrombie and Heaysman [1953] showed a signi-
cant inverse relationship between the speed of movement
of a cell and the number of other cells with which it was
in contact during the observed movement. The decrease
in the cell migration that we observed in the intermediate
area of the repairing cultures could therefore be related to
an increase in the proliferation rate of these cells. The
decrease in cell migration at the end of the wound repair
process could be related to a contact inhibition of
movement occurring when junction has been established
between opposing sheets of cells, as earlier described by
Abercrombie and Heaysman [1954].
Leading cells, migrating toward the wound, are
polarized in that they extend lamellipodia only along the
free edge of the wound. This phenomenon has been
described as contact-stimulated migration by Thomas
and Yamada [1992] and could give direction to the
migratory path. The movement of cells in a given
direction has been related to the fact that they do not
usually move in a direction that will take them over the
surface of another cell, but they are free to move in any
other direction. In a wounded area, cells will conse-
quently be released from contact inhibition of locomotion
and move toward the denuded area [Abercrombie and
Heaysman, 1970]. An important feature to consider is the
highly dynamic formation of lamellipodia at the leading
edge of the cells located at the front of the wound.
Protrusion of the cell membrane is coupled to polymeriza-
tion of actin laments at the leading edge. Different
mechanisms for generating protrusive force have been
proposed [Mitchison and Cramer, 1996]. Either motor
proteins could drive protrusion, or actin polymerization
itself could produce force. The protrusion mechanism is
followed by cell adhesion and traction. The leading cells
at the wound edge seem to act as a tractor for the cells
located behind them. Cell-cell interactions and probably
cell-matrix interaction alterations govern the ability of the
cell population to migrate. These mechanisms are depen-
dent on the extracellular matrix which forms the support
for the cells [Roman and McDonald, 1991]. During the
wound repair, cell surface adhesion molecules play a
likely key role by modulating the cell contact with the
extracellular matrix or with neighboring cells. Among
these cell adhesion molecules, the integrins are known to
Cell Proliferation and Migration in Wound Repair 41
be involved in the regulation of cell migration and
proliferation [Albelda, 1991]. We have recently shown
that specic cellular receptors for extracellular matrix are
involved in cell migration during airway epithelium
repair [Herard et al., 1996]. The a5b1 integrin receptor
and its ligand, bronectin, are upregulated in airway
migrating cells and govern the wound repair process. A
characteristic feature of the migratory process observed
in the present in vitro wound model is the convergence of
the migratory path of the cells located around the wound.
The signals governing the uniform directionality of cell
migration during wound repair are not well characterized.
The guidance of motility could be related to chemotaxis,
haptotaxis and contact guidance. Recently, Nishimura et
al. [1996] demonstrated that keratinocytes are able to
migrate towards the negative pole in direct current
electric elds that are of the same magnitude as measured
in vivo near wounds in mammalian skin. This electrically
directed movement of cells suggests that galvanotaxis
may be one of the physiological mechanisms involved in
wound healing.
CONCLUSIONS
Our results clearly show that in response to wound-
ing, respiratory epithelial cells spread, migrate and prolif-
erate, leading to the reestablishment of the barrier func-
tion of the epithelium. The tracking of cells at the wound
edge associated with video microscopy techniques points
out the coordinated and directional migration path of the
cells. The cells located at the wound edge are character-
ized by the highest migration velocity and the lowest
proliferative activity. The nature of the signals involved
in propagating cell replication in the cells located behind
the wound edge and how it might be related to cell
migration is unknown. Propagation of information is
probably an important mechanism to organize coordi-
nated activity within a repairing area, and the study of this
propagation will be the further step in the understanding
of wound repair processes in respiratory epithelial cells.
ACKNOWLEDGMENTS
The authors are indebted to Ms. R. Smith for
reviewing the paper and thank Dr. Hannion (Cinique
Courlancy, Reims) and Dr. Klossek (Hopital Jean Ber-
nard, Poitiers) for providing the nasal polyps. This work
was supported by grants from Association Francaise de
Lutte contre la Mucoviscidose (AFLM), Pole Tech-
nologique Regional GBM, INSERM (Contrat Normalise
dEtude Pilote), and Ministe`re de lEnseignement
Superieur et de la Recherche.
REFERENCES
Abercrombie, M., and Heaysman, J.E.M. (1953): Observations of the
social behaviour of cells in tissue culture. I. Speed of movement
of chick heart broblasts in relation to their mutual contacts.
Exp. Cell. Res. 5:111131.
Abercrombie, M., and Heaysman, J.E.M. (1954): Observations of the
social behaviour of cells in tissue culture. II. Monolayering of
broblasts. Exp. Cell. Res. 6:293306.
Abercrombie, M., and Heaysman, J.E.M. (1970): Contact inhibition in
tissue culture. In Vitro 6:128142.
Albelda, S.M. (1991): Endothelial and epithelial cell adhesion mol-
ecules. Am. J. Respir. Cell Mol. Biol. 4:195203.
Aggarwal, J.K., and Nandhakumar, N. (1988): On the computation of
motion from sequences of images. A review. Proc. IEEE
76:917935.
Buisson, A.C., Gilles, C., Polette, M., Zahm, J.M., Birembaut, P., and
Tournier, J.M. (1996a): Wound repair-induced expression of
stromelysins is associated with the acquisition of a mesenchy-
mal phenotype in human respiratory epithelial cells. Lab. Invest.
74:658669.
Buisson, A.C., Zahm, J.M., Polette, M., Pierrot, D., Bellon, G.,
Puchelle, E., Birembaut, P., and Tournier, J.M. (1996b): Gelatin-
ase B is involved in the in vitro wound repair of human
respiratory epithelium. J. Cell. Physiol. 166:413426.
Chevillard, M., Hinnrasky, J., Pierrot, D., Zahm, J.M., Klossek, J.M.,
and Puchelle, E. (1993): Differentiation of human surface upper
airway epithelial cells in primary culture on a oating collagen
gel. Epith. Cell Biol. 2:1725.
Fletcher, M.J., Warwick, K., and Mitchell, R.J. (1991): The application
of a hybrid tracking algorithm to motion analysis. IEEE
5:8489.
Gordon, R.E., and Lane, B.P. (1976): Regeneration of rat tracheal
epithelium after mechanical injury. II. Restoration of surface
integrity during the early hours after injury. Am. Rev. Respir.
Dis. 113:799807.
Herard, A.L., Pierrot, D., Hinnrasky, J., Kaplan, H., Sheppard, D.,
Puchelle, E., and Zahm, J.M. (1996): Fibronectin and its a5b1
integrin receptor are involved in the wound repair process of the
airway epithelium. Am. J. Physiol. 271 (Lung Cell. Mol.
Physiol. 15):L726L733.
Keenan, K.P., Combs, J.W., and McDowell, E.M. (1982): Regeneration
of hamster tracheal epithelium after mechanical injury. I. Focal
lesions: quantitative morphologic study of cell proliferation.
Virchows Arch. 41:193214.
Lee, Y., McIntire, L.V., and Zygourakis, K. (1994): Analysis of
endothelial cell locomotion: Differential effects of motility and
contact inhibition. Biotechnol. Bioeng. 43:622634.
McDowell, E.M., Becci, P.J., Schurch, W., and Trump, B.F. (1979): The
respiratory epithelium. VII. Epidermoid metaplasia of hamster
tracheal epithelium during regeneration following mechanical
injury. J. Natl. Cancer Inst. 62:9951008.
Mitchison, T.J., and Cramer, L.P. (1996): Actin-based cell motility and
cell locomotion. Cell 84:371379.
Nikula, K.J., Wilson, D.W., Giri, S.N., Plopper, C.G., and Dungworth,
D.L. (1988): The response of the rat tracheal epithelium to
ozone exposure: Injury, adaptation and repair. Am. J. Pathol.
131:373384.
Nishimura, K.Y., Isseroff, R.R., and Nuccitelli, R. (1996): Human
keratinocytes migrate to the negative pole in direct current
electric elds comparable to those measured in mammalian
wounds. J. Cell Sci. 109:199207.
Rangarajan, K., and Shah, M. (1991): Establishing motion correspon-
dance. CVGIP: Image understanding 54:5673.
42 Zahm et al.
Roman, J.R., and MacDonald, J.A. (1991): Cellular processes in lung
repair. Chest 100:245248.
Sethi, I.K., and Jain, R. (1987): Finding trajectories of feature points in
a monocular image sequence. IEEE Transactions 9:5673.
Shimizu, T., Nishihara, M., Kawaguchi, S., and Sakakura, Y. (1994):
Expression of phenotypic markers during regeneration of rat
tracheal epithelium following mechanical injury. Am. J. Respir.
Cell Mol. Biol. 11:8594.
Tatsuka, M., Jinno, S., and Kakunaga, T. (1989): Quantitative measure-
ment of cell motility associated with transformed phenotype.
Jpn. J. Cancer Res. 80:408412.
Thomas, L.A., and Yamada, K.M. (1992): Contact stimulation of cell
migration. J. Cell Sci. 103:12111214.
Uras, S., Girasi, F., Verri, A., and Toore, V. (1988): A computational
approach to motion perception. Biol. Cybern. 60:7987.
Zahm, J.M., Chevillard, M., and Puchelle, E. (1991): Wound repair of
human respiratory epithelium. Am. J. Respir. Cell Mol. Biol.
5:242248.
Zahm, J.M., Pierrot, D., Chevillard, M., and Puchelle, E. (1992):
Dynamics of cell movement during the wound repair of human
surface respiratory epithelium. Biorheology 29:459465.
Zahm, J.M., Pierrot, D., and Puchelle, E. (1993): Epidermal growth
factor promotes wound repair of human respiratory epithelium.
Wound Rep. Reg. 1:175180.
Zahm, J.M., Pierrot, D., and Puchelle, E. (1994): Photodamage induced
by UV illumination on respiratory epithelial cells. In Jouffrey
B., and Colliex C. (eds.): Proceedings of the 13th International
Congress on Electron Microscopy. Les Ulis: Les Editions de
Physique, Vol. 3A, pp. 8990.
Cell Proliferation and Migration in Wound Repair 43