Copyright

In vitro

models

V. MoulinxfE,

G. Castilloux$,

to study
A. JeanlT2,

wound
D. R. Garre13,

Bum Vol. 22, No. 5, pp. 359-362. 1996

Q i996 Elsevier Science Ltd for ISX All rights reserved
Printed in Great Britain
0305~4179/96
$15.00 + 0.00

healing
F. A. AugerlrZ

l&aboratoire de Recherche des Grands BrCIlPsILOEX, Hbpital du Saint-Sacrement, Qu&ec,

chirurgie, Universit& kaval, Sainte-Foy, Canada and 3Centre des Grands Brirlks, H&l-Dieu
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 in 10
per cent of dermal cells, whereas XI-&O per cent of wotind fibroklasts
contairzed this protein in their cytoplasm. The contractile property of
cells was evaltufed using a three-dimensional cell culture model. OW
results show that wound fibmbhsts contract collagen gels during the
first 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 confaining 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 important 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.ndhealing 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 low or no a-SM actin.
The presence of a-SM actin in wound-healing fibroblasts
and their responseto 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 myofibroblasts are responsible for tissue contractiorP. Myofibroblasts are transiently observed during normal wound
repair, their presenceparallels the active wound contrac-

fibroblasts
and L. Germain
Canada, Dbpartement de
de MO&r&al, Montrkal,

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 fibroblastshave 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 functional 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, woundhealing fibroblasts are different from dermal fibroblasts.
Using phase-contrast microscopy, dermal fibroblasts presented well-known morphoIogica1fibroblast features with
an elongated spindle shape. They were small ceils with
clear cytoplasm. Several strains of wound-healing fibroblasts had been obtained from subcutaneously implanted
spongesza.All strains contained star-shaped large cells
with a high cytopIasmic to nuclear ratio, moreover, these
cells possessedstressfibres. 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
(FigureI). All fibroblasts and myofibroblasts contained
vimentin.

Human wound-healing myofibroblasts continued to

express their characteristics during several passagesin
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. Thesepercentages of C&M actin expressing cells in human dermal
fibroblast cultures are in the range reported by Des-

Figure I. ImmunofIuorescent
stainingof humanfibroblastsculturedfrom dermis(a) or wound-healingtissue(b)
with anti-a-Ski actin antibodies. Note that a proportion of wound-healing
fibroblastsare 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-SMactin was
stablefor 10 passagesat 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

100
--t

WHF3-2

-is-

m7-1

Nomal

skin fibmblasts

equivalent

The culture in monolayer is the most widely usedmethod

to study cellsin 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 tissueengineered 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 fibroblasts (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 passages(four to eight) contracted the
tissue-engineeredequivalent 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

0
0

12

6
7
8
TIME (llA%S)

10

11

12

13

14

Figure t. Curves showing the contraction of collagenlattices

by dermal fibroblasts and wound-healing
fibroblasts. Woundhealing 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 collagen gels after 24 h in culture showed that cells were
physically separatedfrom 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-nalfibroblasts, in vitro. Gel contraction is influenced by
cell number, collagen concentration, cell types and culture
conditions such as the presenceof cytokines and growth
factors in the culture medium9*5*17119.
Since collagen
concentration, cell number and culture conditions were
constant in our collagen gels seededwith fibroblastic cells,
the difference in contraction islikely to be due to functional
and morphological differences between fibroblasts and
myofibroblasts. One of these differences was the significant proportion of myofibroblasts containing a-SM actin
in their cytoplasm compared to fibroblasts. This could
suggestthat the percentage of cells expressing ol-SM actin

Moulin et al: Wound healing fibroblasts

in vitro

can be related to wound contraction in the first few days.

According to Ehriichs hypothesis, collagen gel contraction 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. It c& be
suggested that the increased contraction by myofibroblasts reflects their ability to attach to more fibres because
of their larger size. However, this decrease in the contraction, 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 myofibroblasts depends not only on coordinated cellular
contraction as previously thought, but that myofibroblasts can contract as individual cells.

361
Philadelphia:
3

cell contraction

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.

Majno G, GabbianiG, HirschelBJ, Ryan GB, Statkov PR.

Contraction
of granulation
tissue in vitro: similarity to
smooth muscle. Science 2971; 173: 548-550.
5 Rudolph R. Contraction
and the control of contraction
World

shdure
and function. J .hp Med 1972; 135: 719-725.
2 Rudolph R, Van de Berg J, Ehrlich PH. Wound contraction
and scar contracture. In: Cohen IK, Diegelmann RF, Lindblad
WJ, eds,. WouPtd Healing.
Biochemical
and clinica/
aspects.

] Surg

1980;

4: 279-287.

Darby I, Skally 0, Gabbiani G. R-Smooth muscle actin is

transiently expressed by myofibroblasts during experimental wound healing. Lab Invest 1990; 63: 21-29.
7 Garrel DR, Gaudreau P, Zhang L, Reeves1, BrazeauP.

Chronicadministrationof growth hormone-releasing

factor
increases wound

strength and collagen maturation

nulationtissue.] surgRes

1991;

51:

in gra-

297-302.

Diegelmann RF, Lindblad WJ, Cohen IK. A subcutaneous

implar,t for wound healing studies in humans. J Surg Res

RomprCP, Auger FA, GerrnainL et al. Influence of initial

collagenand cellularconcentrationson the final surface area

40:

229-235.

of dermal and skin equivaIents: a Box-Behnken

Vi&
10

Cell Dev

Biol 1990;

26:

analysis. In

983-990.

GermainL, JeanA, Auger FA, Garrel DR. Humanwound

healing fibroblasts have greater contractile properties than

dermal fibroblasts. J Surg Res 1994; 5 7: 268-273.
11 Desmouliere
A, Rubbia-Brandt
L, Abdiu A, Walz T,
Macieira-Coelho
A, Gabbiani G. a-Smooth muscle actin is
expressed in a subpopulation of cultured and cloned fibroblasts and is modulated by y-interferon. Exp Cell Res 1992;
201:

64-73.

12

Schurch W, Seemayer TA, Gabbiani G. Myofibroblasts.

In:
Stemberg SS, ed. Histolo,,for Paholagists.
New York: Raven,

13

Van de Berg JS, Rudolph R, Poolman WL, Disharoon DR.

Comparative growth dynamics and actin concentration
between cultured human myofibrobIasts from granulating
wounds and derrnal fibroblasts from normal skin. Lab Invest

1992;

1989;

p 109.

61:

532-541.

Colige AC, Lambert CA, Nusgens BV, Lapiere CM. Effect of

cell-cell and cell-matrix interactions on the responseof
fibroblasts to EGF in virto. Biohem ] 1992: 285: 215-221.
15 Bell E, Ehrlich PH. Buttle DJ, Nakatsuji T. Living tissue
formed in vitro and accepted as skin-equivalent tissue of full
thickness. Stience 1981; 2x1: 1042-1054.
16 Germain L, Auger FA. Tissue engineered biomaterials:
biological
and mechanical characteristics. In: Wise DL,
14

Trantolo DJ, Altobelli DE, eds. Encyclopedic

Handbook

of

Biomaferials rind &engineering, PurI I?: applications, vol 1. New

York: Marcel Dekker, 1995; pp 699-734.
17 Delvoye I, WiJiquet P Levi+e
JL, Nusgens BV, Lapikre
CM. Measurement of mechanical forces generated by skin
fibroblasts embedded in a three-dimensional
coliagengel.
] hvest

1 Gabbiani G, Hirschel BJ, Ryan GB, Statkov PR, Majno G.

Granulation tissue as a contractile organ. A study of

an in

407-417.
4

Dermatol

1991;

97:

898-902.

Gillery P, Maquart FX, Le Corre K, Kalis 3, Bore1 JP.

Variability in the retraction of collagen lattices by scleroderma fibroblasfs ~ relationship to protein synthesis and
clinicaf data. Clin Erp Dematol
1991; 16: 324-330.
19 Tingstrijm A, Heldin CH, Rubin K. Regulation of fibrobiastmediated collagen gel contraction
by platelet-derived
growth factor, interleukin-lm
and transforming
growth
factor-/?l. J Cell Sci 1992; 102: 315-322.
20 Skalli 0, Schurch W, Seemayer T, Lagace et al. Myofibroblasts from diverse pathologic settings are heterogeneous in
their content of actin isoforrns and intermediate filament
18

References

forces for collagen lattice contraction:

vitro model of wound contraction. &siae Celi 1990; ZZ:

X986;

Several experimental models have been designed to study

wound healing, in&ding subcutaneously implanted chambers 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 healing tissue. However, these in vitro cultures
lack the tissue architecture provided by the surrounding
extracellular matrix. Three-dimensional
models such as
tissue-engineered equivalents provide improved culture
systems allowing functional 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
equivalent.

Saunders, 1992; p. 96.

EhrlichHP, RajaratnamJBM. Cell locomotion forcesversus

Burns: Vol. 22, No. 5, 1996

362

proteins. Lab inuest 1989; 60: 275285.

21 Murray JC, Pinnell SR. Keloids and excessive dermal scarring. In: Cohen IK, Diegelmann RF, Lindblad WJ, eds. Wuund
Healing. Biachemical 6 clinicA aspects. Philadelphia: Saunders,
3992; p 520.
22 Skalli 0, Gabbiani G. The biology of the myofibroblasts:
relationship
to wound contraction
and fibrocontractive
diseases. In: Clark RAF, Henson PM, eds. 2% Mole&u
rind

Award

Cellulu~ Biology of Wotknd Repair. New-York:

Plenum, 1988;

p373.

Paper accepted 14 November

199.5.

Correspotidence should be addressed to: Dr Lucie Gcrmain, Laboratoires des Grands Btiles/LOEX,
HBpital du Saint-Sacrement,
1050 Chemin Sainte-Foy, Qubbec, QC, Canada GxS &A.

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
Director of the Trauma Service, Massachusetts General Hospital, Boston, USA

Faculty and Emeritus

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.