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.

in vitro models to study wound healing fibroblasts

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