Matrix Vol. 13/1993, pp.

441-446
© 1993 by GustavFischer Verlag, Stuttgart· Jena . New York

Hyaluronic Acid Modulates Proliferation, Collagen and

Protein Synthesis of Cultured Fetal Fibroblasts

BRUCE A. MAST, ROBERT F. DIEGELMANN,

THOMAS M. KRUMMEL 1 and I. KELMAN COHEN
The Wound Healing Center of The Division of Plastic and Reconstructive Surgery and The Division of
Pediatric Surgery, Department of Surgery, Medical College of Virginia, Virginia Commonwealth University,
Richmond, VA23298, USA.

Abstract

Fetal wound healing is characterized by minimal inflammation, mild fibroplasia and rapid, but
organized collagen deposition such that scarring is not apparent. The matrices of fetal wounds
differ greatly from adult wounds in that fetal wounds are persistently enriched with hyaluronic
acid (HA). It has been shown that a reduction in fetal rabbit wound HA results in an adult-like
healing response with increased fibroplasia and neovascularization. These observations suggest
that HA can modulate cellular activity in fetal repair. Therefore, this study was designed to define
the effect of HA on fetal fibroblast function. Fibroblasts from the skin of fetal rabbits were
isolated and maintained in culture medium containing either no HA (controls), 1 [lg/ml, 10 [lg/ml
or 100 [lg/ml of HA (n = 6 for each group). Fibroblast proliferation was quantitated by DNA
content in each culture, and collagen and noncollagen protein synthesis were analyzed by
incorporation of [3Hl proline into collagenase-digestible and collagenase-nondigestible protein,
respectively. At all concentrations tested, HA significantly inhibited fetal fibroblast proliferation
(p < 0.02), but stimulated collagen (p < 0.002) and non collagen protein (p < 0.005) synthesis.
These findings provide further evidence that HA affects the function of fetal fibroblasts.
Moreover, this study in conjunction with previous in utero findings suggests that HA may have a
regulatory influence in scarless fetal healing by affecting cellular function during the repair
process.

Key words: fetal wound healing, fibroblast, hyaluronic acid.

Introduction 1971; Krummel et al., 1987; Seibert et al., 1990), with

The cellular and extracellular events responsible for fetal
and adult wound healing are fundamentally different (Mast
et al., 1992 a; c). Adult healing is a fibrotic process in which
scar formation replaces normal tissue as a consequence of
prominent inflammation, fibroplasia and dense, yet poorly
organized collagen deposition (Levenson et al., 1965; Doil-
Ion et al., 1985). Conversely, fetal wounds are charac-
terized by mild inflammation and fibroplasia (Burrington,
1 Current Address: Division of Pediatric Surgery, Milton S. Her-
shey Medical Center, Hershey, PA
rapid and highly organized collagen deposition (Adzick et
al., 1985; Longaker et al., 1990; Burd et al., 1990) such that
adult-like scarring does not occur (Krummel et al., 1987;
Hallock et al., 1987). In further contrast to adult healing,
the extracellular matrix of fetal wounds contains a persis-
tent abundance of the glycosaminoglycan hyaluronic acid
(HA) (DePalma et al., 1989; Chiu et al., 1990). In adult
wounds HA is primarily a transient component of the very
early wound matrix, where it is thought to bind with fibrin
and thereby provide a scaffold-like matrix which facilitates
inflammatory cell influx (Weigel et al., 1986; 1988).
Hyaluronic acid is also prevalent in tissues undergoing
442 B. A. Mast et al.
true, amphibian regeneration (Toole and Gross, 1971;
Toole and Trelstad, 1971) wherein changes in the level of
HA have been shown to affect cellular proliferation and the
orderly deposition of structural matrix (Toole, 1976).
Based on the absence of scarring in regeneration and fetal
repair, it has been postulated that the persistent abundance
of HA in fetal wounds may have a regulatory effect on
cellular and matrix events such that fetal repair is more
regenerative rather than fibrotic (DePalma et aI., 1989;
Mast et aI., 1991 a). In support of this concept, we have
demonstrated that a significant reduction in the HA content
of fetal rabbit wounds results in an adult-like healing
response with marked increases in fibroplasia, collagen
deposition and neovascularization (Mast et aI., 1992 b).
Therefore, it is hypothesized that HA may be a modulator
of fibroblast function and matrix deposition during fetal
wound healing.
The present study was designed to further analyze the
role of HA as a modulator of fetal healing. Based on the
effect of HA on fibroplasia and collagen deposition
observed in earlier in utero studies (Mast et aI., 1992 b), the
potential modulation of fetal fibroblast function was
studied in a controlled in vitro model. Accordingly, the
influence of HA on fetal dermal fibroblast collagen and
non collagen protein synthesis, as well as proliferation was
analyzed in cell culture.
Materials and Methods
Fibroblast isolation
Fibroblasts were isolated from the skin of fetal rabbits
delivered by Caesarian section on the 24th day of gestation
(term is 31 days). This day of gestation was chosen because
all previous in vivo fetal wound studies performed by our
group were initiated at this time during fetal development.
The fetuses were immediately sacrificed by decapitation
and full thickness skin was removed from the dorsum,
excluding the dorsal fat pad. The skin was finely minced
and incubated at 37°C in 25 ml of 0.2% bacterial colla-
genase (CLSPA grade, Worthington Biochemical Corp.,
Freehold, NJ) solution in Dulbecco's modified Eagle's
medium containing no fetal calf serum (DMEM-O). Max-
imum cellular release was achieved after one and one half
hours of incubation in collagenase. The suspension was
centrifuged at 400 x g for 10 min and the used collagenase
solution discarded. The skin and cells were resuspended in
25 ml of 0.1 % trypsin: 0.1 % EDTA in calcium and mag-
nesium free-Hanks' buffer and incubated for 10 min at
37°C. The released cells were separated from the skin by
filtering the suspension through sterile gauze. The cells
were pelleted by centrifugation at 400 x g for 10 min and
the used trypsinlEDTA solution discarded. The pelleted
cells were resuspended in 25 ml of DMEM, buffered to
pH 7.4 with 25 mM Tricine, supplemented with 10% FCS
and 1% penicillin/streptomycin (DMEM-lO). To deter-
mine the quantity of cells isolated, a 200!l1 aliquot of the
cell suspension was mixed with an equal volume of 1 %
acetic acid to lyse erythrocytes and then the cells were
counted using a hemocytometer. Cell viability was greater
than 90% as determined by Trypan blue exclusion. Cells
were then plated at a density of 4 X 105 cells/well in 24-well
tissue culture plates; the diameter of each well is 16 mm.
The cultures were maintained in a 37°C, room air,
humidified incubator. The viability of the cultures and the
absence of microbial infection were assessed daily by
microscopy. By 24 hours, cell attachment had occurred and
the only cells present in these cultures were fibroblasts. The
DMEM-10 was aspirated and appropriate test medium (see
below) was changed daily.
Determination of proliferation, collagen and
noncollagen protein synthesis
The effect of HA on fetal fibroblasts was evaluated by
culturing cells in medium supplemented with HA (bacte-
rially fermented HA having a molecular weight of 1 to 1.8
million daltons containing less than 0.1 % protein; gift of
Genzyme, Inc, Cambridge, MA). Cultures were maintained
in DMEM-lO with either no HA (controls), 1 !lg/ml of HA
(groupA), lO!lg/ml of HA (groupB) or lOO!lg/ml of HA
(group C). These concentrations are comparable with those
found in vivo in fetal rabbit wounds (DePalma et aI., 1989).
The HA-supplemented medium was added following cell
attachment (18 to 24 h after plating) and changed daily.
The cultures were maintained in these media for 72 h. Pro-
liferation, collagen synthesis and noncollagen protein pro-
duction were determined using the microassay technique
previously described by our laboratory (Diegelmann et aI.,
1990). Briefly, after 72 h of HA exposure, the medium was
removed from each culture well and the cells were pulsed
for five hours with fresh DMEM-O (0.5 ml) containing
20 !lCi of tritiated proline and 0.1 mM ascorbate. Collagen
synthesis was determined as a function of radioactivity
incorporated into collagenase-digestible protein. Noncolla-
gen protein synthesis was determined by incorporation of
radiolabeled proline into collagenase-nondigestible pro-
tein. The effect of HA on cell proliferation was determined
by quantitation of DNA contained in each culture well at
the end of the study period. DNA measurement was
achieved using a spectrophotofluorometric Hoechst dye
binding assay (Labarca and Paigen, 1980). Collagen pro-
duction was calculated as collagen synthesized per nano-
gram of DNA present in the culture (absolute collagen
synthesis or "ACS") or as a percent of total protein synthe-
sized (relative collagen synthesis or "RCS").
 Hyaluronate Modulates Fetal Fibrobla~t Function 443

Statistical analysis

Statistically significant effects of HA were detected by

student t test comparison of the mean values obtained from
each HA culture group to that of the control group (n = 5 or
greater). All values are expressed as a mean +/- standard
deviation. ·p<0.005

Results
DNA quantitation
Fibroblasts grown in medium containing no HA (con- Controls 1 l1g!ml 10 ~ml 100 ~ml
trols) had 5343 +/- 310ngofDNAperwell (n = 6). DNA n:5 n:6 n:6 n:5
contained in group A was 4665 +/- 200 (n = 6); DNA Hyaluronic Acid
contained in groupB was 4634 +/- 246 (n = 6); DNA
contained in group C was 4588 +/- 505 (n = 6). Com- Fig. 2. The effect of hyaluronic acid on noncollagen protein syn-
thesis by fetal fibroblasts. Cultures exposed to hyaluronic acid
pared to the control group, there was significantly less demonstrated a significant increase in synthesis of noncollagen
DNA (p < 0.02) in each of the cultures exposed to HA protein, thus representing a stimulatory effect of hyaluronic acid
(groups A, B, C) (Figure 1). However, there was no differ- on noncollagen protein synthesis. (All values are mean +/- stan-
ence among the three test groups. dard deviation).

Noncollagen protein synthesis 1~~---------------------------'

Noncollagen protein synthesis by control cultures was *T

163.95 +/- 13.71 dpm/ng DNA (n = 5); group A produc- •
tion was 217.43 +/- 26.17 (n = 6); groupB production
was 255.55 +/- 30.41 (n = 6); groupC production was
252.22 +/- 48.11 (n = 5). Therefore HA exposure resulted
·p<0.002
in a significant increase in non collagen protein synthesis
(p <0.005) (Figure 2).

6OCO~--------------------------~
-r • Controls 1 ~ml 10 ~ml 100 ~ml

5000
• * n:6 n=6 n=6 n=5
Hyaluronic Acid
4000

l 3000
Fig. 3. The effect of hyaluronic acid on absolute collagen synthesis
(collagen production per ng DNA contained in the culture). Treat-
ment of fetal fibroblasts with hyaluronic acid resulted in approxi-
~
Q
·p<0.02
mately a two-fold increase in absolute collagen synthesis. Thus, it
2(0) appears that hyaluronic acid stimulates the production of collagen
by these cells. (All values are mean +/- standard deviation).
Hxx)

o
Controls 1 ~ml 10 l1g!ml 100 ~ml
Collagen synthesis
n:6 n:6 n:6 n:6 Absolute collagen synthesis (ACS) in the control culture
Hyaluronic Acid was 61.58 +/- 4.75 dpm/ng DNA (n = 6); ACS for
group A was 105.38 +/- 10.95 (n = 6); ACS for groupB
Fig. 1. The effect of hyaluronic acid on proliferation of fetal fibro- was 115.58 +/- 13.39 (n = 6); ACS for group C was 98.16
blasts. The quantity of DNA contained in a culture is directly pro-
portional to proliferation. Cultures of fibroblasts exposed to
+/- 17.27 (n = 5). ACS was significantly increased (p
hyaluronic acid contained significantly less DNA than control < 0.002) by treating fibroblasts with HA (Figure 3).
cultures, thus representing an inhibitory effect of hyaluronic acid The control culture group demonstrated a relative colla-
on proliferation. (All values are mean +/- standard deviation). gen synthesis (RCS) of 6.40 +/- 0.37% (n = 5); RCS for
 444 B. A. Mast et al.
10
* tained in HA-treated cultures compared to controls. This is
~ not a cytotoxic effect of HA because the metabolic activity
*
J
8 of the fibroblasts actually was stimulated by HA; fibro-
6 gen protein synthesis as well as absolute collagen synthesis.
j
*p<O.OOl
4
~
:a 2 was not observed at the highest concentration of HA tested
~ because relative collagen synthesis was unchanged in this
Controls 1 j.LgIml 10 j.LgIml 100 j.LgIml
n=5 n=6 n=6 n=5
Hyaluronic Acid
Fig. 4. The effect of hyaluronic acid on relative collagen synthesis
(collagen synthesis as a percent of total protein synthesis). Treat-
ment of fetal fibroblasts with low concentrations (l!-tg/ml or
10 !-tg/ml) of hyaluronic acid resulted in a significant increase in
relative collagen synthesis. However, fibroblasts exposed to treat-
ment with 100 !-tg/ml of hyaluronic acid demonstrated no change
in relative collagen synthesis compared to controls. Therefore, it
appears that at the higher concentration of hyaluronic acid, colla-
gen and noncollagen protein synthesis were stimulated to similar
degrees. (All values are mean +/- standard deviation).
group Awas 8.30 +/- 0.75% (n = 6); RCS for group B was
7.68 +/- 0.23% (n = 6); RCS for groupC was 6.70 +/-
0.31 % (n = 5). The RCS of groups A and B was signifi-
cantly greater than controls (p < 0.001), although the RCS
for group C was similar to the control group (p = 0.219)
(Figure 4).
Discussion
The significance of the persistently high content of HA in
fetal wounds has been highly speculative. It has been
demonstrated recently that a 50% reduction in the HA
content of fetal wounds is associated with marked increases
in fibroplasia, collagen deposition and neovascularization,
such that the healing response becomes more adult-like
(Mast et aI., 1992 b). Thus, it appears that HA influences
the cellular and/or extracellular events in fetal healing. The
present cell culture study was undertaken to more specifi-
cally define the effect of HA on fibroblast function. Dermal
fibroblasts were studied because it is this population of cells
from which "wound" fibroblasts arise, and these are the
cells that would be affected by the high levels of fetal wound
HA present in the repair process. Consequently, the con-
centrations of HA used in this study are comparable to the
concentrations found in fetal rabbit wounds (DePalma et
aI., 1989).
Hyaluronic acid inhibited fibroblast proliferation as
shown by the significantly reduced quantities of DNA con-
blasts exposed to HA had significantly increased noncolla-
The increase in relative collagen synthesis seen with the
lower concentrations of HA indicated that HA stimulated
collagen synthesis to a greater degree than noncollagen
protein synthesis. The greater effect on collagen synthesis
group.
The mechanism by which HA affects the activity of cul-
tured fetal fibroblasts remains undefined. Hyaluronic acid
has been observed to form a pericellular coat attached to
the surface of cells (Underhill and Toole, 1979; Knudson
and Toole, 1985), a process thought to be mediated by cell
surface receptors (Underhill and Toole, 1980; 1981;
McCary et aI., 1989; Yoneda et aI., 1990). A pericellular
coat of HA would create an anionic boundary around the
fibroblasts that could result in the attraction or localization
of cationic growth factors and thus provide the fibroblasts
with a greater accessibility to growth factors. Fibroblasts
produce transforming growth factor-B (TGF-B) (Pierce et
aI., 1988), so that a pericellular HA boundary may prevent
loss of this cytokine into the culture medium. The signifi-
cance of such an interaction between HA and growth fac-
tors is implied by the observations that TGF-B stimulates
collagen production by fibroblasts (Roberts et aI., 1986)
and has caused increased collagen deposition in fetal rabbit
wounds (Krummel et aI., 1988). Alternatively, HA may
alter fibroblast function as a direct result of cell surface
binding. Hyaluronic acid has been shown to promote pro-
tein tyrosine phosphorylation and phospholipid break-
down in a heart fibroblast culture model. This suggests that
second messenger generation may occur as a consequence
ofHA-cell surface binding (Turley, 1989) and thus provide
intracellular signals for alterations in cellular function.
The inhibition of cultured fetal fibroblast proliferation
by HA is consistent with earlier in vivo observations
because a reduction in the HA content of fetal rabbit
wounds (removal of a proliferation inhibitor) results in
increased fibroplasia. However, the finding that HA stimu-
lated fetal fibroblast collagen production in culture seems
to be contrary to animal studies in which removal of HA
from fetal rabbit wounds results in increased collagen
deposition (Mast et aI., 1992 b). In the animal studies, HA
was removed from fetal wounds by treatment with
hyaluronidase, thus not only reducing HA content, but
generating HA degradation products. These degradation
products have been shown to stimulate angiogenesis and
collagen deposition in fetal wounds (Mast et aI., 1991 b).
The present study provides a controlled analysis of the
effect of high molecular weight HA on fibroblast function
and does not address the potential interactive biologic

activities of HA and its degradation products. Such interac-
tion may have a combined effect on actual collagen deposi-
tion in the fetal wound.
This study provides further evidence that in contrast to
adult healing, fetal wound HA is more than a structural
component of the extracellular matrix. The observations
that HA inhibited proliferation, but stimulated collagen
and noncollagen protein synthesis by fetal fibroblasts sug-
gest that HA modulates cellular activity in fetal repair.
Indeed, the persistent abundance of HA may have a regula-
tory influence in scarless fetal healing by modulating cellu-
lar function during the repair process.
The ultimate goal in studying fetal wound healing is to
understand the mechanisms of this scarless process and
hopefully apply these mechanisms to pathologic healing in
children and adults. Such modulation of abnormal healing
would have the potential to provide great benefit to these
patients with conditions that are often associated with sig-
nificant morbidity and mortality.
Acknowledgements
This study was supported by: NIH Grants GM 41343, GM
20298, GM 47566 and Plastic Surgery Educational Foundation
Research Grant. Work contained in this manuscript was presented
at: American Association of Plastic Surgeons, San Antonio, TX,
April 1991.
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