in vitro models to study wound healing fibroblasts
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Bum Vol. 22, No. 5, pp. 359-362. 1996
Copyright Q i996 E lsevier Sci ence Ltd for ISX All rights reserved
Printed in Great Britain
0305~4179/9 6 15.00 + 0.00
In vitro models to study wound healing fibroblasts
V. MoulinxfE, G. Castilloux$, A. JeanlT2, D. R. Garre13, F. A. AugerlrZ and L. Germain
l&aboratoire de Recherche des Grands BrCIlPsILOEX, Hbpital du Saint-Sacrement, Qu&ec, Canada, Dbpartement de
chirurgie, Universit& kaval, Sainte-Foy, Canada and 3Centre des Grands Brirlks, H&l-Dieu de MO&r&al, Montrkal,
Cmada
Phenotypic and contractile properties of hltmanfibroblastsfrom dermis
and from cm expuimental wound model were studied in vitro . When
ctiltured in monolayer, dermal fibroblasts had an elongafed spindle
shape, were smull in diameter and grew at a high rate. Wound
fibroblask grew slowly rind were large, star shaped and had
cytcplasmic stiessfibres. Smooth muscle alpha adin was detecfed n 10
per cent of dermal cells, whereas XI-&O per cent ofwotind fibroklasts
contairzed this protein in their cy toplasm. The contractile property of
cells was eval tufed using a three-dimensional cell culture model. OW
results show that wound fibmbhsts contract collagen gels during the
firs t days more stror;gly than dermal jibrubhsfs. These results show
that, irr vitro, ,woupld fibroblusfs have greafer contractile capacity fkan
den& cells. The significant proporiiorz of wourld fibrobhsfs confain-
ing E-smooth mu& actin suggests fhaf a-smooth muscle nctin mtio
may be reiafed to wound conkacfion. Copyright 0 1996 Eisevier
Science Ltd for ISBI.
Burns, Vol. 2.2, No. 5 359-362, 1996
Introduction
Contraction and granulation tissue formation are impor-
tant steps for the healing of a skin wound. During these
processes,specialized mesenchymal cells play a critical
role. These cells, so-called myofibroblasts pr wou.nd-
healing fibroblasts, have morphoiogical and biochemical
features between those of the fibroblast and of the smooth
mu&e (SM) cells. The hallmark of these cells is the
expression of actin isoform present in SM cells, the c(-SM
actin, in a large quantity in opposition to dermal fibroblasts
that have been shown to express ow or no a-SM actin.
The presence of a-SM actin in wound-healing fibroblasts
and their response o SM pharmacological stimuli1suggest
a role of these cells in wound contraction.
The mechanism of tissue contraction during wound
healing is not completely understqod. Two main theories
have been proposed to explain this process. The first
theory suggests that fibroblast locomotion within the
connective tissue induces wound contraction. In this
model, it is the fibroblasts, assingle units, that generate the
forces necessary for contraction by reorganizing the
extracellular matrix3. In the second theory, the myofibro-
blasts are responsible for tissue contractiorP. Myofibro-
blasts are transiently observed during normal wound
repair, their presenceparallels the active wound contrac-
tion phase.Once contraction has stopped, myofibroblasts
are no longer detected in normal scarring. The hypothesis
of the second mechanismproposes that forces generated
by myofibroblasts are transmitted to ether cells and
surrounding connective tissue through their gap junctions
and basementmembrane.Thus, the myofibroblasts would
act asa multicellular unit to contract the tissuez.
In order to understand the physiological events
involved in human wound healing, wound-healing fibro-
blastshave been cultured from human granulation tissue7,8.
They were compared to fibroblasts from human dermal
skin9 using two in vitro culture systems. Cultures in
monolayer were used to study the phenotype and growth
of fibroblasts from various donors. To analyse the func-
tional aspect, cells were seededwithin coiIagen gels and
used to compare contractile properties of cells cdtured
from dermis and wound-healing tissue.
Culture in monolayer
When cultured on plastic tissue culture dishes, wound-
healing fibroblasts are different from dermal fibroblasts.
Using phase-contrast microscopy, dermal fibroblasts pre-
sented well-known morphoIogica1 ibroblast features with
an elongated spindle shape. They were small ceils with
clear cytoplasm. Several strains of wound-healing fibro-
blasts had been obtained from subcutaneously implanted
spongesza.All strains contained star-shaped arge cells
with a high cytopIasmic to nuclear ratio, moreover, these
cells possessed tress ibres. Growth curves showed that
myofibroblasts always grew more slowly than fibroblasts.
To better characterize the phenotype of cells cultured
from wound-healing tissue, these cultures were labelled
for a-SM actin. According to myofibrobiast lines, the
proportion of cells expressing a-SM actin varied between
20 and 80 per cent. In contrast, this smooth muscle cell
marker was detected in 10 per cent of dermal fibroblasts
(Figure ). All fibroblasts and myofibroblasts contained
vimentin.
Human wound-healing myofibroblasts continued to
express their characteristics during several passages n
culture. Their growth rate was significantly lower than
fibroblasls, even after 10 passages,suggesting that the
population of slower growing myofibroblasts was not
contaminated by faster dividing fibroblasts. Thesepercent-
ages of C&M actin expressing cells in human dermal
fibroblast cultures are in the range reported by Des-
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Figure I. ImmunofIuorescenttainingof human ibroblasts ultured rom dermis a) or wound-healingissue b)
with anti-a-Ski actin antibodies. Note that a proportion of wound-healing fibroblasts re strongly positive for
a-SM actin (a), whereas most of the normal dermal fibroblasts are negative (b). ( x 275.)
moulieres et a .
(7-20
per cent on the 4th passage).
Percentage of myofibroblasts containing ok-SM actin was
stable or 10 passages t least and was significantIy greater
in all instances than in fibroblasts. Similar observations
were reported for myofbroblasts obtained from rat and
human wound healing tissue, Dupuytrens diseaseand
fibrocontractile disease12*13.
Tissue engineered equivalent
The culture in monolayer is the most widely usedmethod
to study cells n vitro. This technique allows analysisof the
morphology of the cells and measurementof parameters
like growth rate or protein synthesis. However, cells
cultured in monolayer are not in a physiological state. In
vivo, sells are surrounded with extracellular matrix which
is known to
have
many actions in
cell morphology and
functions14.Dermal and wound-healing skin fibroblasts are
confined in a tissu.ewhere 80 per cent of matrix proteins are
collagen. To study the functional aspects of dermal or
wound-healing fibroblasts, we used Bells modelIs which
consistsof cultmed cells in a collagen lattice. This method
allows the study of cell contractile properties in an in
vivo-like environment through tissue engineering of
human wound-healing equivalents.
Dermal cells were seeded in bovine type I collagen
solution and poured into plastic bacteriological petri dishes
according to the method of Rompre et a1.9The tissue-
engineered equivalents were cultured during 14 days and
the capacity of contraction was evaluated by measureof
the equivalent diameter.
The tissue-engineered equivalent contraction rate is
dependent on cell number and collagen concentration16.
Therefore, these parameters were established to obtain a
gradual contraction of the equivalents by dermal fibro-
blasts (reduction by 50 per cent of the initial surface area
after 5 days). After 14 days in culture, the equivalents
produced with dermal fibroblasts were contracted to less
than 20 per cent of their initial surfacearea (F@re 2 ).
Cells isolated from human wound-healing tissue and
cultured for a few passagesfour to eight) contracted the
tissue-engineered quivalent more rapidly (reduction of 20
to 50 per cent of the initial surface area after 24 h) than
dermal fibroblasts (reduction of 25 per cent of the initial
surfaceareaafter 24 h). A greater extent of contraction was
measured in gel containing wound-healing fibroblasts
during the first day of culture then contraction was much
100
--t WHF3-2
-is- m7-1
4 Nomal skin fibmbla sts
0
0 12 3 4 5 6 7 8 9 10 11 12 13 14
TIME (llA%S)
Figure t. Curves showing the contraction of collagen attices
by dermal fibroblasts
and
wound-healing fibroblasts. Wound-
healing fibroblasts from five healthy volunteers (WI-IF Z-I, 3-2,
4-1,5-l and 7-1) were cultured from subcutaneously implanted
PVA sponges. Note that the extent of contraction is greater
when wound-healing fibroblasts are used compared with dermal
fibroblasts. (Mean of duplicate k s.d. II= 3.)
slower. The final surface area was similar for both cell
populations after 14 days of culture (QWE 2). Histological
analysis of wound-healing fibroblasts that populated col-
lagen gels after 24 h in culture showed that cells were
physically separated rom one another. After 14 days in
culture, a time at which most of the contraction had
occurred, ceilswere stiI1scattered throughout the collagen
gel. Somecellswere alsopresent at the surfaceof the gello.
These results show that wound fibroblasts from human
granulation tissue have greater contractile capacity than
den-nal ibroblasts, n vitro. Gel contraction is nfluenced by
cell number, collagen concentration, cell types and culture
conditions such as the presenceof cytokines and growth
factors in the culture medium9*5*17119.ince collagen
concentration, cell number and culture conditions were
constant in our collagen gels seededwith fibroblastic cells,
the difference in contraction is ikely to be due to functional
and morphological differences between fibroblasts and
myofibroblasts. One of these differences was the signifi-
cant proportion of myofibroblasts containing a-SM actin
in their cytoplasm compared to fibroblasts. This could
suggest hat the percentage of cells expressing ol-SM actin
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Moulin et al: Wound healing fibroblasts in vitro 361
can be related to wound contraction in the first few days.
According to Ehriichs hypothesis, collagen gel contrac-
tion by fibroblasts is a consequence of their locomotion3
through the collagen matrix. Cells become intimately
linked to the fibrils and pull on themlo. This locomotion
process cou d explain the regular contraction of the gel by
dermal fibroblasts during the l&day period. I t c& be
suggested that the increased contraction by myofibro-
blasts reflects their ability to attach to more fibres because
of their larger size. However, this decrease in the contrac-
tion, rate compared to fibroblasts after the first days
suggest that they move much slower. The collagen gel
contraction occurring within the first 24 h, a time at which
cells are physically separated in the ge5 is likely to be due
to cell-matrix linkages and not to cell-cell contacts.
Therefore, we hypothesize that wound CIosure by myo-
fibroblasts depends not only on coordinated cellu lar
contraction as previously thought, but that myofibro-
blasts can contract as individual cells.
Several experimental models have been designed to study
wound heal ing, in&d ing subcutaneously implanted cham-
bers or sponges in which fibroblasts migrate and collagen
is deposited 7*8,18 In the present study, this system was
used to obtain cells from human wound-healing tissue to
ctilture them
Monolayer cultures are useful to compare phenotypic
and growth properties of human fibroblasts from dermis
and wound heal ing tissue. However, these in vitro cultures
lack the tissue architecture provided by the surrounding
extracellu lar matrix. Three-dimensional models such as
tissue-engineered equivalents provide improved culture
systems allowing funct ional studies (e.g. contraction).
They could be used to study the cellular biology of normal
wound-heaiing and the abnormal healing process leading
to hypertrophic scars, myofibroblasts being also present in
human hypertrophic scars and other fibrocontractile
diseaseszu-:2. Work is in progress to evaluate the effects of
various cytokines and eventually drugs on wound repair
using this tissue-engineered human wound-healing equi-
valent.
Acknowledgements
The authors acknowledge Claude Marin for photographic
assistance. This study was supported by the Fondation des
Pompiers du Quebec pour les Grands Bn%s, Fondation
de lH6pital du Saint-Sacrement and Reseau des grands
brirles du Fends de ia recherche en santC du Quebec. L.G.,
F.A.A. and D.G. were recipients of Scholarships from
FRSQ.
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Paper accepted 14 November 199.5.
Correspotidence should be addressed to: Dr Lucie Gcrmain, Labora-
toires des Grands Btiles/LOEX, HBpital du Saint-Sacrement,
1050 Chemin Sainte-Foy, Qubbec, QC, Canada GxS &A.
Award of the G. Whitaker International
Burns Prize for
1996,
Palermo, Italy
During a
meeting
held on April 26th 19% at the seat of the G. Whitaker Foundation, Pakermo, Italy,
after examining scientific activities in the fields of research, teaching, clinical organization, prevention
and co-operation among the nations, presented by various candidates. The Adjudicating Committee
unanimously decided to award the prize for 19% to:
John Burke MD Emeritus Professor of Surgery at the Harvard University Medical Faculty and Emeritus
Director of the Trauma Service, Massachusetts General Hospital, Boston, USA
The prize is awarded for the following:
for dedicating a lifetime to teaching and to the assistance of patients in the sector of the surgery. His
vast and qualified activity in the field of burns, to our knowledge of which he has contributed with
numerous publications on various aspects, particularjy infections and metabolism, His studies for the
realization of artificial skin and the use of biomaterials have been notable.
The official prize-giving of the prestigious award will be held on September 26th 19% in Palermo at
the seat of the G. Whitaker Foundation in the presence of the authorities and of representatives of the
academic, scientific and cultural world.