Original Contribution

Journal of Cosmetic Dermatology, 12, 36--40

Comparison of noncross-linked and cross-linked hyaluronic acid

with regard to efficacy of the proliferative activity of cutaneous
fibroblasts and keratinocytes in vitro
Johannes Wohlrab, MD,1 David Wohlrab, MD,2 & Reinhard H. H. Neubert, PhD3
1

Department of Dermatology and Venereology, Halle (Saale), Germany

Department of Orthopedic Surgery, Halle (Saale), Germany
3
Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany
2

Summary

Background Intradermal application of hyaluronic acid (HA) in varying chain length

and cross-linking density is used routinely for hydrodynamic volume replacement of
the extracellular matrix to reduce the clinical effects of aging.
Objectives In vitro data show that via receptors of the hyaladherin group hyaluronic
acid has additionally direct or indirect effects on cells. In the case of native noncrosslinked HA, it has been proved that the proliferative and metabolic activity of
cutaneous fibroblasts can be increased. The aim of this study was to investigate
whether these effects can be proved also for cross-linked HA and how these effects
can be quantified for different preparations.
Materials and Methods The effect on proliferative activity in cultures of native cutaneous
fibroblasts and keratinocytes was investigated for noncross-linked HA, for noncrosslinked HA with added glycerol, for HA that was stabilized in the carboxyl and hydroxyl
groups per inner esterification, and for HA that was chemically cross-linked by 1,4butanediol-diglycidylether, mixed in small particles in a biphasic compound with native
HA, each in different concentrations (0.1, 1.0 and 10.0 mg/mL).
Results HA that was stabilized in the carboxyl and hydroxyl groups per inner
esterification induces the strongest proliferative effect on both cell types. Native
noncross-linked HA and chemically cross-linked HA show a rather modest
proliferative effect and on fibroblasts only, whereas noncross-linked HA with added
glycerol in high concentrations provokes a rather antiproliferative effect.
Conclusions The data show that HA does induce direct effects on cells depending on
type and density of the cross-linkage. The practical relevance in terms of a metabolic
filler effect needs to be verified in clinical studies.
Keywords: hyaluronic acid, fibroblast, keratinocyte, proliferation

Introduction
Correspondence: Johannes Wohlrab, MD, PhD, Department of
Dermatology and Venereology, Martin Luther University
Halle-Wittenberg, Ernst-Kromayer-Strasse 5; D-06097 Halle (Saale),
Germany. E-mail: johannes.wohlrab@medizin.uni-halle.de
Accepted for publication October 17, 2012

36

Hyaluronic acid (HA) and its salts or deprotonized forms

(hyaluronates) are linear structured and comparatively
long biopolymers.1 Balazs introduced hyaluronan as
collective term for the acid and its poly-anions that
occur within physiological pH conditions.2

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Effects of hyaluronic acid on fibroblasts and keratinocytes

HA consists of a large number of basic saccharide units

linked in a linear chain by glycosidic bonds: C14H21NO11
with a molecular weight of 3 793 208 g/mol, and the
disaccharidic head- and endgroup C14H22NO11 and
C14H22NO12.3 The number of the basic units can reach
10.000 and more. The molar mass of the HA of
vertebrates would be approximately 4 million Da. The
overall structure based on all intra- and intermolecular
interactions of the HA molecules is also referred to as
supramolecular structure.4
Within physiological conditions, HA is detectable
both in a free dissolved form as well as chemically
bound in almost all organs of the human body. Because
of its large hydrodynamic volume, HA is a substantial
component of the extracellular matrix (ECM) of the skin,
mainly of the corium. It is the basic substance where
cell can proliferate, differentiate, and migrate, and has
direct and indirect effects on the cellmatrix as well as
cellcell interactions.5 Depending on its chain length,
HA has also direct regulative effects via intra- and
extracellular and membrane-embedded receptors (hyaladherins).69 Therefore, HA works as a signaling molecule for the regulation of inflammatory processes,10,11
tumorgenesis,12 metastasis formation,13 neoangiogenesis14 and wound healing.15 Very important here are the
products of the biodegradation of HA, resulting from
enzymatic hydrolysis (hyaluronidases)16 or from elimination.17,18 Their functional groups are less masked
and therefore show an increased physiological activity.
In the course of extrinsic aging, especially due to
UVB radiation, defects within the matrix metalloproteinase 1 cause the proteolytic cleavage of dermal collagen.19 The reduced synthesis of HA causes
alterations that result in a modified dermal morphology15,20,21 with a reduced turgor pressure within the
tissue and the formation of wrinkles. In response to
this, the intradermal application of nonanimal (produced by fermentation) stabilized HA (NASHA) in
varying chain length and cross-linking density is a
common procedure to replace the hydrodynamic volume of the extracellular matrix and thus to reduce the
clinical effects of aging.22 In addition, it has been
proved that native HA has pro-proliferating effects on
cutaneous fibroblasts.23 The expression of CD44 seems
to have a great impact here,24 although it is not clear
whether the effects are caused by HA or rather by its
products of biodegradation.25 There is also a debate on
whether HA has an indirect influence on the metabolism of the fibroblasts and their collagen production via
biomechanical stretching26 or cytokine cascades.27 The
pro-proliferative effects of HA have been proved for
noncross-linked HA only.28 It is still unclear whether

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. J. Wohlrab et al.

these effects can be applied also for cross-linked HA

and whether these effects are dependent on the type of
cross-linkage resp. stabilization. Therefore, various HA
produced by fermentation from established cosmetic
preparations for intradermal application have been
comparatively tested in vitro in this study regarding
their impact on the proliferative activity of fibroblasts
and keratinocytes.

Materials and methods

Materials

For the tests, sterile HA produced by fermentation from

commercially available derma filler preparations were
used: native noncross-linked HA (Hyal System, Merz
Pharmaceutical GmbH, Frankfurt, Germany), native
noncross-linked HA with added glycerol (Mesolis Plus,
Anteis S.A., Genf, Switzerland), HA that was stabilized
in the carboxyl and hydroxyl groups per inner esterification (Hyal ACP, Merz Pharmaceutical GmbH,
Frankfurt, Germany), and HA that was chemically
cross-linked by 1,4-butanediol-diglycidylether, mixed in
small particles in a biphasic compound with native HA
(Restylane Vital, Q-Med AB, Uppsala, Sweden). In
addition, glycerol (Sigma-Aldrich, purity  99%, St.
Louis, USA) was tested for comparison. The different
concentrations to be tested in vitro were prepared under
sterile conditions by dilution of the original preparations
with cell medium with a ratio of 0.1, 1.0, and 10.0 mg/
mL, respectively. Every individual HA test preparation
as well as glycerol in different concentrations was tested
against a nontreatment control. Supplementary, the
concentration with the least effect of both the native
noncross-linked HA as well as of the HA that was stabilized in the carboxyl and hydroxyl groups per inner
esterification were tested in combination.
Methods

The incorporation of [3H]-thymidin in the DNA was

used as a marker for the proliferative activity of the
tested cellular systems. The cellular systems were
native cutaneous fibroblasts and native keratinocytes
from surgical specimens of juvenile human foreskin.
The cells were isolated according to current standard.
The cells were cultivated in 24er microtiter plates
(Greiner Bio-One GmbH, Frickenhausen, Germany) at
37 C, 20% CO2 and 80% humidity until confluency of
85% was reached and then exposed to the test preparations for 24 h. Subsequently, 0,5 lCi [3H]-thymidin
(Hartmann Analytic GmbH, Braunschweig, Germany)

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Effects of hyaluronic acid on fibroblasts and keratinocytes

. J. Wohlrab et al.

per sample was added. After 1 h the cells were harvested using a cell harvester (Inotech, Wohlen, Switzerland), transferred into scintillator tubes (Ultima
Gold, PerkinElmer, Rodgan, Germany) and after further
24 h measured in a liquid scintillation counter (Wallac-ADL GmbH, Freiburg, Germany). For each test setting six cultures were tested independently (n = 6).
Biometric evaluation

The statistical software SigmaStat V 1.0 for Windows

was used for biometric evaluation. The statistics were
descriptive and were calculated from the results of all
independent tests. The box-plot charts show the 5th,
25th, 50th (median), 75th, and the 95th percentile,
respectively. After checking the normal distribution
and the variance homogeneity, the independent samples were compared using one-way analysis of variance, the StudentNewmanKeuls test or Bonferroni
test, and the KruskalWallis test. At a significance level
of P < 0,05 differences were assessed as statistically
significant. Significant differences compared with the
control were indicated with *.

Results
The results for fibroblasts and keratinocytes for each
preparation, dependent on the respective concentration

as well as including the combination of native noncross-linked HA and stabilized HA, each in concentration with the least effect, are shown in Figures 1 and 2.

Discussion
The data show that direct effects of HA on the proliferative activity of fibroblasts and keratinocytes are in vitro
principally verifiable. However, these effects seem to be
dependent on the bioavailability of certain binding
domains of the HA. Multiple influences need to be considered here. Besides the type and density of cross-linkage, products of biodegradation are most likely involved
in initiating effect cascades. Also, a different expression
of individual hyaladherins that depends on the cell type
and possibly also on the level of differentiation of cells
needs to be considered. The studies presented here do
not allow substantiated statements hereto.
Nevertheless, it is evident that besides the wellknown hygroscopic effects of intradermal applied HA,
other effects do appear by the induction of the proliferative activity of fibroblasts especially. To what extend
these effects will have a practical relevance in terms of
increasing the collagen synthesis can only be speculated. Based on data given by Fisher et al. 200826 and
Rock et al. 201027 we can assume that the induction
of proliferative activity of cutaneous fibroblasts cause
clinical relevant metabolic changes within the dermal

Figure 1 Box-plot chart of [3H]-thymidin incorporation used a marker for the proliferative activity of native human cutaneous fibroblasts (NHDF) as n-fold of control after 24 h incubation time (n = 6). A = 0.1 mg/mL; B = 1.0 mg/mL; C = 10.0 mg/mL; D = 0.1 mg/
mL HSYS and 0.1 mg/mL HACP; CONT = control; HSYS = Hyal System; HACP = Hyal ACP; COMB = combination of Hyal System
and Hyal ACP; RESV = Restylane Vital; MESP = Mesolis Plus; GLYC = Glycerol; * = statistical significant difference to control
(P  0.05).

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Effects of hyaluronic acid on fibroblasts and keratinocytes

. J. Wohlrab et al.

NHKC

Figure 2 Box-plot chart of [3H]-thymidin incorporation used a marker for the proliferative activity of native human keratinocytes (NHKC)
as n-fold of control after 24-h incubation time (n = 6). A = 0.1 mg/mL; B = 1.0 mg/mL; C = 10.0 mg/mL; D = 0.1 mg/mL HSYS and
0.1 mg/mL HACP; CONT = control; HSYS = Hyal System; HACP = Hyal ACP; COMB = combination of Hyal System and Hyal ACP;
RESV = Restylane Vital; MESP = Mesolis Plus; GLYC = Glycerol; * = statistical significant difference to control (P  0.05).

connective tissue. Clinical studies are necessary to

prove, whether and to what extend a metabolic activation of fibroblasts sets in. Based on the available in vitro data, in which commonly intradermal applied
concentrations of HA were used, a clinical relevance of
these effects can be regarded in the first days after
application as very likely.
Besides the common application of HA as a temporary dermal volume replacement filler, another field of
application could open up: a metabolic filler function
for the longer term.
Further studies are needed to allow substantiated
statements for individual preparations. For the preparations tested in this study, it can be said that HA that
was stabilized in the carboxyl and hydroxyl groups per
inner esterification may be suited to provide this metabolic filler effect.

Acknowledgments
The authors thank Mrs. Sylke Fasshauer and Mrs.
Claudia Bruhne for excellent technical assistance. The
study was fully sponsored by Merz Pharma GmbH,
Frankfurt, Germany.

Conflict of interests
JW received lecture fees by Merz Pharma GmbH. DW
and RH declare no conflict of interest.

2013 Wiley Periodicals, Inc.

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