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Modulation of Inflammation by Cicaderma Ointment
Accelerates Skin Wound Healing
Christophe Morin, Audrey Roumegous, Gilles Carpentier, Ve´ ronique Barbier-Chassefie` re,
Laure Garrigue-Antar, Ste´ phane Caredda, and Jose´ Courty
Laboratoire Croissance Cellulaire, Re´ paration, et Re´ge´ne´ ration Tissulaires (CRRET), E
´
quipe d’Accueil Conventionne´ e, Centre
National de la Recherche Scientifique 7149, Universite´ Paris-Est Cre´ teil, Cre´ teil, France (C.M., A.R., G.C., V.B.-C., L.G.-A.,
J.C.); and Laboratoires Boiron, Sainte Foy-le` s-Lyon, France (S.C.)
Received October 4, 2011; accepted June 27, 2012
ABSTRACT
Skin wound healing is a natural and intricate process that takes
place after injury, involving different sequential phases such as
hemostasis, inflammatory phase, proliferative phase, and re-
modeling that are associated with complex biochemical events.
The interruption or failure of wound healing leads to chronic
nonhealing wounds or fibrosis-associated diseases constitut-
ing a major health problem where, unfortunately, medicines are
not very effective. The objective of this study was to evaluate
the capacity of Cicaderma ointment (Boiron, Lyon, France) to
accelerate ulcer closure without fibrosis and investigate wound
healing dynamic processes. We used a necrotic ulcer model in
mice induced by intradermal doxorubicin injection, and after 11
days, when the ulcer area was maximal, we applied Vaseline
petroleum jelly or Cicaderma every 2 days. Topical application
of Cicaderma allowed a rapid recovery of mature epidermal
structure, a more compact and organized dermis and collagen
bundles compared with the Vaseline group. Furthermore, the
expression of numerous cytokines/molecules in the ulcer was
increased 11 days after doxorubicin injection compared with
healthy skin. Cicaderma rapidly reduced the level of proinflam-
matory cytokines, mainly tumor necrosis factor (TNF)-
␣
and oth-
ers of the TNF pathway, which can be correlated to a decrease
of polymorphonuclear recruitment. It is noteworthy that the
modulation of inflammation through TNF-
␣
, macrophage in-
flammatory protein-1
␣
, interleukin (IL)-12, IL-4, and macro-
phage– colony-stimulating factor was maintained 9 days after
the first ointment application, facilitating the wound closure
without affecting angiogenesis. These cytokines seem to be
potential targets for therapeutic approaches in chronic wounds.
Our results confirm the use of Cicaderma for accelerating skin
wound healing and open new avenues for sequential treat-
ments to improve healing.
Introduction
Skin wound healing involves a series of complex processes
that need the interaction of cytokines and growth factors
produced by many different specialized cells. During normal
wound healing, these orderly events can be classified in four
overlapping phases: inflammation, formation of granulation
tissue, re-epithelialization, and matrix formation/remodel-
ing. When these stages are delayed for more than a few
weeks, wound consequently heals unusually slowly, such as
in diabetic foot ulcer (Jeffcoate and Harding, 2003) or more
generally necrotic ulcer (Disa et al., 1998). This defines clin-
ically the chronic wound, one of the most common disorders,
which severely impairs the quality of life of the patient and
creates a huge financial burden on the healthcare system.
Classically caused by a variety of events such as trauma,
exposure to heat, cold, corrosive material, or radiations, and
problems with blood circulation, skin ulcers are open wounds
often accompanied by the sloughing of inflamed tissue. To
improve this situation, many different therapeutic ap-
proaches have been tested to accelerate healing processes
without fibrosis in the scar. For example, in several animal
models for wound repair, a significant increase of healing
was obtained by topic application of growth factors such as
keratinocyte growth factor (Shannon et al., 2006; Henemyre-
Harris et al., 2008), basic fibroblast growth factor (Akita et
al., 2008), transforming growth factor (Cho et al., 2010), and
platelet-derived growth factor (Yan et al., 2011). However,
costs and side effects restricted the use of these compounds
and opened the way for new approaches like the use of
This work was supported by the Laboratoires Boiron of France.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
http://dx.doi.org/10.1124/jpet.111.188599.
ABBREVIATIONS: MMP, matrix metalloproteinase; PMN, polymorphonuclear neutrophil; TNF, tumor necrosis factor; sTNF, soluble TNF; IL,
interleukin; MIP, macrophage inflammatory protein; M-CSF, macrophage– colony-stimulating factor; GM-CSF, granulocyte macrophage–colony-
stimulating factor; G-CSF, granulocyte– colony-stimulating factor; IFN, interferon; SDF-1, stromal cell-derived factor-1; TCA-1, T cell activation-1;
TIMP, tissue inhibitor of metalloproteinase.
1521-0103/12/3431-115–124$25.00
T
HE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 343, No. 1
Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics 188599/3793692
JPET 343:115–124, 2012
115
protectors or co-receptors of these growth factors such as
mimetics of endogenous sulfated glycosaminoglycans (Gar-
cia-Filipe et al., 2007; Barbier-Chassefie`re et al., 2009).
Since antiquity, plant extracts such as St. John’s wort
(Hypericum perforatum L.) have been used to treat wounds
within folk medicine in various countries. However, in the
majority of cases, the composition and the scientific evidence
of their efficiency remain to be established. It is noteworthy
that Cicaderma ointment (Boiron, Lyon, France) is marketed
mainly in Europe and has been used since the middle of the
20th century in the treatment of wounds, superficial burns of
limited extent, and insect bites. This ointment is prepared by
the extraction of fresh Calendula officinalis L., H. perforatum
L., and Achilea millefolium L. aerial parts in Vaseline mixed
with the hydroalcoholic extract of Ledum palustre L. Sepa-
rately, some of these extracts, which are known to inhibit in-
flammation processes and matrix metalloproteinase (MMP) ac-
tivity (Oztu¨ rk et al., 2007; Dell’Aica et al., 2007a,b; Su¨ ntar et al.,
2011), are traditionally used to improve wound healing.
To better understand the claimed use of Cicaderma in
wound healing and identify new therapeutic targets, we
tested its effects in a model of necrotic skin ulcer healing that
reproduces human chronic wound (Barbier-Chassefie`re et
al., 2009) and investigated the dynamic processes implicated
in wound healing. For the first time, the expression levels of
40 cytokines/molecules involved in the inflammation pro-
cesses were analyzed when the ulcer reached its maximal
area and during wound healing.
Materials and Methods
Animal Model of Skin Ulceration. Housing of animals and
anesthesia were performed following the guidelines established by
the Institutional Animal Welfare committee with the European
guide for care and use of laboratory animals. Standardized skin
ulceration was performed by intradermal doxorubicin (Doxorubicin
Teva 0.2%) injection on the shaved dorsum of male Swiss mice
(Janvier, Le Genest-St-Isle, France) as described previously (Bar-
bier-Chassefie`re et al., 2009). In brief, animals were anesthetized
intraperitoneally with sodium pentobarbital (Ce´va Sante´ Animale,
Libourne, France). The backs of the mice were shaved with a hair
clipper and depilated with Veet depilatory cream (Reckitt Benckiser,
Massy, France). Two days after depilation, mice received 150 lofa
2 mg/ml doxorubicin solution by intradermal injection on the depil-
ated area. The maximum of skin ulcer area was reached 11 days
after doxorubicin injection. That day (day 1) was the first day of
treatment with Cicaderma or Vaseline (the main excipient of Cica-
derma), which were then applied topically every 2 days on the ulcer.
Ulcers were photographed every 2 days (days 1, 3, 5, 8, 10, 12, 14, 17,
19, and 21) and cleaned until their complete closure. The lesion size
was measured three times by using ImageJ software (http://imagej.
nih.gov/ij/) for each ulcer, and the mean was calculated. Biological
samples for histological analysis were taken from sacrificed animals
on days 1, 3, 5, 10, 17, 19, and 30 after the first application of the
ointment, whereas biochemical analysis was done on days 1, 5, and 10.
Histological Studies. Skin samples were fixed in formaldehyde-
buffered solution and embedded in paraffin wax. Serial 8-m sec-
tions were prepared. Staining with Masson trichrome was used to
study the skin regeneration of five mice in each experimental group
(Junqueira et al., 1979). For collagen deposition study, tissue sam-
ples were stained with sirius red, and pictures were taken by using
a Leitz Laborlux 12 PolS microscope under polarized light. For
inflammatory cell studies, sections were prepared as above and
stained with May-Grunwald-Giemsa. Polymorphonuclear neutrophil
(PMN) blue spots (⫻250) were counted in three sections per mouse
with three mice in each group (Barbier-Chassefie`re et al., 2009). For
blood-vessel density evaluation, paraffin-embedded sections of skin
samples on days 3, 5, and 10 after doxorubicin injection were rehy-
drated by using a decreasing percentage alcohol series as described
previously (Barbier-Chassefière et al. 2009). Anti-CD31 primary an-
tibody (1: 25 dilution; BD Biosciences, San Jose, CA) was incubated
at room temperature for 2 h and washed in 3% bovine serum albu-
min/phosphate-buffered saline. Then sections were incubated with a
second antibody-fluorescein isothiocyanate (anti-rat IgG; 1:100 dilu-
tion; Jackson ImmunoResearch Laboratories Inc., West Grove, PA)
for2hatroom temperature, and after three washes they were
mounted with Vectashield mounting medium (Vector Laboratories,
Burlingame, CA) (Erba et al., 2011). Specific fluorescence intensity
was evaluated by using the TECAN Infinite M1000 plate reader
(Tecan, Durham, NC). The blood vessel density corresponded to the
fluorescence of a section labeled by CD-31 antibody minus the auto-
fluorescence of this section without CD-31 labeling of three sections
per mouse (n ⫽ 3 mice).
Biochemical Studies. Proteins were extracted from skin sample
following the manufacturer’s instructions with slight modifications.
Skin biopsies were minced and incubated in the sample diluent
buffer (Quantibody Mouse Inflammation Array 1; Raybiotech, Nor-
cross, GA) for1hat37°C before homogenization by using a Potter-
Elvehjem glass-Teflon homogenizer. The homogenate was centri-
fuged for 5 min at 13,000g to remove debris and insoluble material.
Aliquots of the supernatant were assayed for total protein content by
the bicinchoninic acid method or stored at ⫺80°C until analysis.
These protein extracts (200 g/ml) were used with Quantibody
Mouse Inflammation Array 1 (Raybiotech) to quantify 40 cytokines in
the kinetics of wound healing skin subjected to Vaseline or Cicaderma
treatment. The binding of each cytokine on the membrane was revealed
by autoradiography and quantified by the Protein Array Analyzer for
ImageJ program developed for ImageJ software (http://rsb.info.nih.gov/
ij/macros/toolsets/Protein%20Array%20Analyzer.txt). Each assay was
performed in duplicate from three mice per experimental group for each
day tested.
Statistical Analysis. All results reported are the mean (⫾
S.E.M.) of independent determinations. Differences between the
means in two groups were evaluated by using Student’s paired t test;
p values ⬍0.05 were considered significant.
Results
Ulcer Area Studies
Intradermal injection of doxorubicin was reported previ-
ously to induce a necrotic skin ulcer, which regenerates spon-
taneously in mice and constitutes a good model to study the
first phases of skin wound healing (Barbier-Chassefie`re et
al., 2009). As shown in Fig. 1, the measurement of the ulcer
area indicates that topical applications of Vaseline from days
1 to 21 did not modify the kinetics of ulcer closure compared
with untreated skin. However, topical treatment with Ci-
caderma ointment induced an acceleration of healing com-
pared with the Vaseline-treated group by reducing the
surface area of ulcer by 20 to 25% in the first week of
treatment. It is noteworthy that Cicaderma application
significantly induced the complete closure of ulcer 2 days
earlier than what was observed with Vaseline. Macro-
scopic observations indicated that ulcers treated with Ci-
caderma appeared less inflamed, less red, and less thick
than those treated with Vaseline (Fig. 2).
Histological Studies
Dermal Reconstruction. The effects of Cicaderma appli-
cation on dermal reconstruction have been assessed by his-
116 Morin et al.
tological studies using Masson staining throughout ulcer clo-
sure. On day 1, corresponding to the maximum of the ulcer
area, the total destruction of the epidermis and dermis was
clearly observed compared with the mature healthy skin
(Fig. 3, a and b). Then the different steps of the healing
processes were followed daily. Major modifications including
epidermal cell proliferation, illustrated by thickening edges
throughout the ulcer, were noticed in both Cicaderma- and
Vaseline treated-mice. It is noteworthy that these effects
occurred from day 2, just after one topical application of the
ointment and were still persistent on day 5 (Fig. 3, c-f). On
day 10, the reconstitution of organized skin layers seemed to
proceed for the animals treated with Cicaderma, and the
thick skin still reflected an important activation (Fig. 3h). In
contrast, Vaseline induced the production of new tissue in
the dermis and hypodermis, but its organization seemed
more anarchic (Fig. 3g), suggesting the development of fibro-
sis in these animals. These differences between the two treat-
ments were still observed on day 17, mainly in layers below
the epidermis that seemed better organized in Cicaderma-
treated animals (Fig. 3, i and j). This was confirmed by the
organization of collagen (stained in green in Fig. 3), which
was less pronounced in sections treated with Vaseline than
in those treated with Cicaderma. Finally, at day 29 a greater
thickness of the epidermis was seen in Vaseline-treated an-
imals compared with Cicaderma-treated animals (Fig. 3, k
and l). Regarding the number of epithelial cell layers, a faster
maturation of epidermal structures was induced by Cicad-
erma. Moreover, the compact and organized dermis in the
healing zone exhibited more common features with normal
skin than the one observed with Vaseline treatment.
Collagen Organization. The histological studies also al-
lowed us to compare collagen organization in the dermis
treated with Vaseline or Cicaderma by sirius red staining,
which was visualized by polarized light. The collagen net-
work was studied on the edges of the ulcer at early stages of
wound healing (days 1, 3, and 5) or at later stages, inside the
healing ulcer area. When the ulcer reached its maximal area
(Fig. 4a), collagen fibers displayed heterogeneity and ap-
peared fragmented and with longer and disorganized fibers
with horizontal and vertical crossing alongside. This was
probably caused by the normal degradation of these fibers by
proteases in the ulceration processes. On day 3, collagen was
more abundant, without any organization between fibers in
Vaseline-treated ulcers (Fig. 4b). I n contrast, treatment
with Cicaderma induced the presence of better defined
intertwining fibers, reflecting the initiation of structural
organization of collagen bundles (Fig. 4c). This was con-
firmed distinctly on days 5 and 10 (Fig. 4, d and f versus e
and g). At these times, collagen fibers of Cicaderma-
treated ulcers were slim and well defined with clear inter-
lacements that reflected organization of these fibers,
whereas the crosswise organization of collagen bundles
was not so apparent for Vaseline treatment. When ulcers
were finally closed, the collagen organization started to
appear in Vaseline-treated animals, whereas in those
treated with Cicaderma an increased amount of fibers and
a better organization in collagen network were observed
(Fig. 4, h and j versus i and k).
Polymorphonuclear Infiltration. Inflammation was
evaluated through the measurement of PMN recruitment at
the edges of the ulcer (Fig. 5a). The number of PMNs was
high when the ulcer reached its maximal area (day 1) and
decreased by 2-fold on day 3 after the first treatment with
Vaseline. In contrast, the topical application of Cicaderma
maintained the high number of PMNs in the wound. On day
5, PMNs increased in the Vaseline-treated group at the level
of day 1 and diminished by 30% on day 10. Cicaderma treat-
ment allowed the progressive inhibition of PMN recruitment
until day 10, at levels significantly lower than with Vaseline
Fig. 1. Effect of Cicaderma on ulcer size. Eleven days after doxorubicin
injection (day 0), Vaseline (dashed line) or Cicaderma (solid line) were
applied topically to the ulcers every 2 days. The surface area of each ulcer
was measured as described under Materials and Methods and reported at
each time point as the percentage of the surface area at baseline (day 1).
Ulcer areas of animals without any treatment are represented by small
dotted line. Each result is the mean (⫾ S.E.M.) of three independent
determinations in each of 10 mice. Statistical analysis was performed by
using Student’s paired t test. ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001.
Fig. 2. Macroscopic aspect of ulcer
wound healing. Eleven days after
doxorubicin injection (day 1), Vaseline
or Cicaderma were applied topically to
the ulcers every 2 days (D).
Cicaderma’s Effect on Inflammation and Skin Wound Healing 117
treatment, suggesting a reduction of inflammatory processes
in the latter steps of wound healing.
Angiogenesis. CD31 staining was performed to analyze
microvessel formation during the angiogenesis phase of
wound healing (Vecchi et al., 1994). Blood vessel density was
not modified by either treatment, but significantly decreased
at day 10 compared with days 3 and 5 (Fig. 5b).
Biochemical Studies
Inflammatory Status at the Maximal Ulcer Area. Be-
fore evaluating whether Cicaderma modulated the inflam-
matory response during the wound healing process, we first
measured the level of 40 inflammatory cytokines/molecules
in skin ulcer samples when the ulcer reached its maximal
area (day 1) and compared it with normal skin (Fig. 6).
Eleven days after doxorubicin intradermal injection (day 1),
the majority of molecules (34/40) were significantly increased
in the nontreated ulcer, except GM-CSF, IFN-␥, IL-12p70,
SDF-1, TCA-3, and TIMP-2 whose levels remained un-
changed. Just before the first application of the ointment, the
TIMP-1 level was remarkably increased by 10 times, whereas
B-lymphocyte chemoattractant (BLC), CD30-L, eotaxin, eo-
taxin-2, Fas-L, fractalkine, G-CSF, IL-3, IL-6, IL-12p40p70,
keratinocyte chemoattractant (KC), macrophage inflamma-
tory protein (MIP)-1␣, MIP-1␥, sTNF RI, and sTNF RII were
augmented by more than 2-fold. Other important molecules
involved in wound healing, such as TNF-␣, IL-4, and IL-10,
rose from 100 to 200%.
Effect of Cicaderma and Vaseline Treatments. Based
on these observations obtained on day 1, we compared the
levels of all cytokines in ulcers treated with Vaseline or
Cicaderma on days 5 and 0 after induction by doxorubicin.
Overall, in comparison with Vaseline treatment, the levels of
the majority of these cytokines/molecules synthesized in the
ulcer were reduced by Cicaderma at days 5 and/or 10.
At day 5 (Fig. 7a), Cicaderma significantly reduced the
amount of molecules involved in the TNF pathway such as
TNF-␣, sTNF RI, and sTNF RII by 24, 43, and 35%, re-
spectively. Moreover, this treatment decreased classic pro-
inflammatory cytokines/molecules such as IFN-␥, IL-2, IL-
12p40p70, IL-12p70, MIP-1␣, and MIP-1␥.Wewere
surprised to find that other known molecules involved in
wound healing such as IL-1␣, IL-1, or GM-CSF were not
significantly modified by the two treatments (data not
shown). It is noteworthy that the amounts of some anti-
inflammatory cytokines were also reduced by topical ap-
plication of the ointment. Cicaderma reduced IL-4 level by
23% and maintained a stable level of IL-10 4 days after the
first application while Vaseline increased IL-10 by approx-
imately 40% (Fig. 7a).
Fig. 3. Histological effects of Vaseline and Cicaderma treat-
ment on skin ulcers. Tissue samples were stained with
Masson trichrome as described under Materials and Meth-
ods. a Mature healthy skin. b, ulcer 11 days after doxoru-
bicin intradermal injection. c, e, g, i, and k, Vaseline-
treated ulcer after 2, 4, 9, 16, and 29 days of treatment (D3,
D5, D10, D17, and D30), respectively. d, f, h, j, and l,
Cicaderma-treated ulcer after 2, 4, 9, 16, and 29 days of
treatment (D3, D5, D10, D17, and D30), respectively. Bars,
2.0 mm.
118 Morin et al.
G-CSF and M-CSF, described as hematopoietic mole-
cules, were significantly reduced by Cicaderma, whereas
their amount was not influenced by Vaseline compared
with day 1 (Fig. 7a). Moreover, the level of the metallopro-
teinase inhibitors TIMP-1 and TIMP-2 involved in colla-
gens and extracellular matrix degradation were reduced
after Cicaderma treatment by 68 and 33%, respectively.
Finally, the detection of Fas ligand, which is involved in
the regulation of apoptotic cell death, was also diminished
by 50% in Cicaderma-treated ulcer compared with the
Vaseline group (Fig. 7a).
On day 10, the healing processes continued, and we noticed
that there were no statistical differences between the two treat-
ments for the majority of proteins (27/40) (Fig. 7b). However,
the expression of 13 cytokines/molecules was significantly de-
creased by 15 to 35% by Cicaderma compared with Vaseline
treatment (Fig. 7b). Cicaderma was able to significantly reduce
the level of the proinflammatory cytokines fractalkine, IL-
12p70, IL-17, MIP-1␣, and TNF-␣, but also the anti-inflamma-
tory cytokines (IL-4 and IL-13) or other molecules (MCP-1,
M-CSF, lymphotactin, Rantes, SDF-1, and TIMP-2). The
amount of proinflammatory cytokines IL-17 and Rantes was
Fig. 4. Histological effects of Vaseline and Cicaderma treat-
ment on skin ulcers. Tissue samples were stained with
sirius red as described under Materials and Methods. Rep-
resentative pictures from the area corresponding to the
ulcer edges (days 1, 3, and 5) or inside the healing ulcer
area at later stages (days 10, 17, and 21) are shown. a, ulcer
11 days after doxorubicin intradermal injection. b, d, f, and
h, Vaseline-treated ulcer after 2, 4, 9, and 16 days of treat-
ment (D3, D5, D10, and D17), respectively. c, e, g, and i,
Cicaderma-treated ulcer after 2, 4, 9, and 16 days of treat-
ment (D3, D5, D10, and D17), respectively. Bars, 100 m.
Cicaderma’s Effect on Inflammation and Skin Wound Healing 119
reduced by 33 and 35%, respectively (Fig. 7b). The hematopoi-
etic molecules MCP-1 and SDF-1 were decreased by 29 and
26%, while the anti-inflammatory cytokine IL-13 was main-
tained stable close to the level observed on day 1 in Cicaderma-
treated ulcer, preventing the increase induced by treatment
with Vaseline (Fig. 7b). Lastly, Vaseline application increased
TIMP-2 by 25%, whereas Cicaderma reduced the level signifi-
cantly by 18% (Fig. 7b).
It is noteworthy that five molecules involved in inflamma-
tion (IL-12p70, IL-4, M-CSF, MIP-1␣, and TNF-␣) were sig-
nificantly diminished on both days 5 and 10 (Fig. 8). There-
fore, Cicaderma seemed to modulate the inflammation
during wound healing by maintaining a reduced level of
these specific factors compared with Vaseline. As presented
in the histological studies on day 5 (Figs. 3, e and f and 4, d
and e) and day 10 (Figs. 3, g and h and 4, f and g), the
regulation of these five molecules probably was implicated in
epidermal-cell proliferation, better collagen organization,
and quicker re-epithelialization of the skin. As for day 5,
TNF-␣, the major cytokine involved in inflammation, was
maintained stable by Cicaderma treatment, whereas the
Vaseline-treated ulcer induced a higher level than at day 1
(Fig. 8a). However, the level of TNF-␣ with Cicaderma re-
mained higher than in healthy skin. Whereas the levels of
M-CSF and IL12p70 in Vaseline-treated ulcer remained un-
changed from day 1 and close to those observed in normal
skin (Fig. 8, b and c), Cicaderma reduced significantly the
concentration of these cytokines at a lower level than on day
1 and in healthy skin. It is noteworthy that Cicaderma re-
duced the level of IL-4 from the first application at a level
close to the one of healthy skin, whereas the IL-4 level re-
mained equivalent to day 1 and higher than in normal skin
after Vaseline application (Fig. 8d). The concentration of
MIP-1␣ in Cicaderma-treated ulcer was significantly lower
than the one observed with Vaseline treatment but it re-
mained higher than in normal skin (Fig. 8e).
Discussion
Nonhealing wounds remain a major health problem, and
new treatments are required to accelerate ulcer closure. Us-
ing a mouse model of skin ulcer induced by intradermal
Fig. 5. a, leukocyte recruitment at the ulcer edges. Vaseline and Cicaderma treatment at days 3, 5, and 10 were established after May-Grunwald coloration
of paraffin sections. Inflammatory cells were counted in 10 independent fields of three sections of three mice of each experimental group. Results are
expressed as the mean ⫾ S.E.M. b, for blood vessel density evaluation, paraffin-embedded sections of skin samples at days 3, 5, and 10 after doxorubicin were
used. Specific fluorescence intensity linked to anti-CD31 primary antibody was evaluated by using the TECAN Infinite M1000 plate reader. The blood vessel
density corresponded to the fluorescence of a section labeled by CD-31 antibody minus the autofluorescence of this section without CD-31 labeling of three
sections from three mice. Results are expressed as the mean ⫾ S.E.M. Statistical analysis was performed by using Student’s paired t test. ⴱ, p ⬍ 0.05; ⴱⴱ,
p ⬍0.01; ⴱⴱⴱ, p ⬍ 0.001.
Fig. 6. Cytokine modifications at the maximum of the ulcer
area (day 1). Cytokines were extracted as described under
Materials and Methods, and their quantification was done
by using Quantibody Mouse Inflammation Array 1 from
Raybiotech. Results are expressed as percentage of cyto-
kine level in normal skin and represent the mean ⫾ S.E.M.
of three animals in duplicate. Statistical analysis was per-
formed by using Student’s paired t test. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍
0.01; ⴱⴱⴱ, p ⬍ 0.001.
120 Morin et al.
injection of doxorubicin (Barbier-Chassefie`re et al., 2009),
the kinetics of the ulcer closure were studied in the presence
of Cicaderma ointment. To focus on skin wound healing pro-
cesses, topic treatment with only Cicaderma started at day 1
when the ulcer reached its maximal area. This treatment is
compared with the classic clinical treatment using petroleum
jelly (Vaseline), which provides a protective moist environ-
ment that facilitates re-epithelialization and wound healing
and is classified by the Food and Drug Administration as a
skin protectant (Food and Drug Administration, HHS, 2003).
The Hemostasis/Inflammation Phases. Eleven days af-
ter doxorubicin injection, the area of the ulcer was maximal.
Cell damage, blood vessel injury, and degradation of the
collagen network induced by many toxic effects of doxorubi-
cin, including the production of an important oxidative
stress, led to clot formation. This clot is considered as an
important reservoir of molecules such as cytokines, growth
factors that are involved in the chemoattraction of various
cells during the early steps of healing (Frank et al., 2000;
Marin et al., 2001). In our ulcer model, 11 days after doxo-
rubicin injection, the expression of cytokines/molecules was
completely modified compared with healthy skin (Fig. 5).
Among the numerous molecules classically involved in the
early steps of hemostasis/inflammation phases of wound
healing, Rantes, MCP-1, MIP-1␣, MIP-1␥, eotaxin, and frac-
talkine were significantly increased, which correlates with
the recruitment of inflammatory cells in the wound bed, as
demonstrated by PMN counting (Fig. 5a). PMNs are de-
scribed as a major source of proinflammatory cytokines IL-
1␣, IL-1, IL-6, and TNF-␣, which are overexpressed in our
ulcer model, to stimulate newly attracted monocytes to dif-
ferentiate into M1 macrophages. These proinflammatory cy-
tokines can also be released by endothelial cells and periph-
eral blood monocytes in response to thrombin stimulation
(Mahdavian Delavary et al., 2011). In addition to these in-
flammatory cytokines, we noticed a significant 4-fold in-
crease in M-CSF, a hematopoietic cytokine described as an
important chemoattractant for PMNs.
Effects of Cicaderma on the Granulation Phase. It is
noteworthy that from its first topical applications (days 3 and
5) Cicaderma induced a significant acceleration of ulcer clo-
sure as shown by the macroscopic measurement of the ulcer
area. The effect of Cicaderma allowed not only the recovery of
a mature epidermal structure, a more compact and organized
dermis, but also a rapid improvement of the collagen bundle
organization close to mature healthy skin. Differences ob-
served in the collagen fiber network can be notably related to
H. perforatum L., which has been described as an inhibitor of
MMP-2 and MMP-9 activities (Dell’Aica et al., 2007a). H.
perforatum L. down-regulates the expression of both MMPs
through the inhibition of the extracellular signal-regulated
kinase 1/2 signaling pathway (Dona` et al., 2004). In addition,
the activation of the fibroblasts and their increased collagen
production by H. perforatum L. (Oztu¨ rk et al., 2007)
strengthen the beneficial effects of Cicaderma in the granu-
lation phase of wound healing. H. perforatum L. was also
described to reduce in vivo the recruitment of PMNs attach-
ing to the vascular endothelium at the site of injury, leading
to a diminution of inflammation and angiogenesis (Dell’Aica
et al., 2007b). This can be linked to the decrease of PMNs,
considered as inflammatory markers, in the wound bed of
Cicaderma-treated ulcers. This reduction in the PMN num-
ber is not only mirrored by the decrease of hematopoietic
cytokines levels such as G-CSF and M-CSF, but also by the
reduction of MIP-1␣ and TNF-␣ levels, in accordance with
the improvement of the granulation phase seen in our histo-
logical studies.
According to the central role of TNF-␣ proposed by Wein-
stein and Kirsner (2010) in the pathogenesis of wound heal-
ing, the reduction in TNF-␣ level associated with the de-
crease of the TNF receptors sTNF RI and sTNF RII induced
by Cicaderma at day 5 is of importance to explain the accel-
Fig. 7. Cytokine levels after treatment by Vaseline or Cicaderma (days 5 and 10). a, cytokines statistically modified after 4 days (day 5) of Cicaderma
treatment. b, GM-CSF level and cytokines statistically modified after 9 days (day 10) of Cicaderma treatment. Cytokines were extracted as described
under Materials and Methods, and their quantification was done by using Quantibody Mouse Inflammation Array 1 from Raybiotech. Results are
expressed as percentage of cytokine level measured at the maximum of the ulcer area (day 1) and represent the mean ⫾ S.E.M. of three animals in
duplicate. Statistical analysis was performed by using Student’s paired t test. ⴱ, p ⬍ 0.05.
Cicaderma’s Effect on Inflammation and Skin Wound Healing 121
eration of ulcer closure. The decrease of TNF-␣ could partic-
ipate in the down-regulation of numerous inflammatory cy-
tokines, i.e., IL-1, IL-6, IL-12, and IL-17, but also the
synthesis of cell surface adhesion molecules involved in ker-
atinocyte proliferation, PMN migration, and adhesion to en-
dothelium (Mahdavian Delavary et al., 2011). In the granu-
lation phase, proinflammatory cytokines such as TNF-␣ and
other molecules of the TNF pathway are produced by macro-
phages/monocytes predominantly recruited by MIP-1␣
(Kondo and Ishida, 2010). However, the reduction of anti-
inflammatory cytokines IL-4 and IL-10 by Cicaderma sug-
gests that this ointment did not simply act as an anti-inflam-
matory system enhancer but as a general modulator
regulating both proinflammatory and anti-inflammatory pro-
cesses such as TNF-␣ (Liechty et al., 2000; Werner and
Grose, 2003). It is noteworthy that C. officinalis L. was able
to reduce the levels of TNF-␣, IFN-␥, and IL-6 in an in vivo
model of inflammation (Preethi et al., 2009) and promote
re-epithelialization in a skin excision model (Preethi and
Kuttan, 2009). Other studies suggested that the specific lack
of endogenous IFN-␥ can reduce IL-12 levels, which signifi-
cantly enhances granulation tissue formation and wound
closure (Ishida et al., 2004). Thus, according to these find-
ings, the beneficial and similar effects of Cicaderma from day
5 on the granulation tissue could be attributed to the pres-
ence of C. officinalis L. in the ointment.
Effects of Cicaderma on the Angiogenesis and Re-
modeling Phases. In our ulcer model, the angiogenesis
phase appeared to begin before day 5 as shown by the label-
ing of endothelial cells (Fig. 6b). On days 3, 5, and 10, angio-
genesis was not modified by Cicaderma treatment, but fur-
ther studies should be used to confirm this finding. However,
Cicaderma modified the pattern of fractalkine, IL-13, IL-17,
lymphotactine, Rantes, and SDF-1 only at day 10, whereas
only IL-4, IL-12, MCP-1, M-CSF, TIMP-2, and TNF-␣ were
decreased on both days 5 and 10. While highlighting the
importance of these cytokines in the regulation of skin wound
healing, these findings suggest that 1) the remodeling phase
of the wound healing process starts between days 5 and 10,
and 2) Cicaderma treatment acts possibly on both the re-
epithelialization and remodeling phases. Furthermore, our
results show for the first time the involvement in skin wound
healing processes of IL-17, an inducer of the production of
many other cytokines (IL-6, G-CSF, GM-CSF, IL-1, trans-
forming growth factor-, and TNF-␣) and chemokines includ-
ing IL-8 and MCP-1 (Akdis, 2010), by fibroblasts, endothelial
Fig. 8. Levels of TNF-␣ (a), M-CSF
(b), IL-12p70 (c), IL- 4 (d), and MIP-1␣
(e) in normal skin, at the maximum of
ulcer area (D1), 4 days (D5), and 9
days (D10) after the first application
of Vaseline or Cicaderma. Cytokines
were extracted as described under
Materials and Methods, and their
quantification was done by using
Quantibody Mouse Inflammation Ar-
ray 1 from Raybiotech. Results are ex-
pressed in percentage of cytokine
level measured at the maximum of
ulcer area (day 1) and represent the
mean ⫾ S.E.M. of three animals in
duplicate. Statistical analysis was
performed by using Student’s paired t
test. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍
0.001 Vaseline versus Cicaderma. #,
p ⬍ 0.05; ##, p ⬍ 0.01; ###, p ⬍ 0.001
compared with normal skin.
122 Morin et al.
or epithelial cells, keratinocytes, or macrophages. Frac-
talkine, which is secreted by monocytes/macrophages and
endothelial cells in response to inflammatory mediators and
oxidative stress, was decreased by Cicaderma treatment.
This could reduce macrophage and fibroblast accumulation
(Ishida et al., 2008), endothelial cell production of vascular
endothelial growth factor (Ryu et al., 2008), and finally an-
giogenesis (Clover et al., 2011). On day 10, because SDF-1 is
proposed to simultaneously promote re-epithelialization and
delay contraction, the reduction of SDF-1 level by Cicaderma
implies the slowing down of the re-epithelialization phase
and the acceleration of wound contraction, which is classi-
cally described to improve skin wound healing in rodents
(Sarkar et al., 2011). On the contrary, the important decrease
of G-CSF level for both treatments suggests the reduction of
wound contraction. Thus, Cicaderma could play an important
role in the improvement of wound healing kinetics by partic-
ipating in the fine-tuning of the contraction regulation. Fur-
thermore, Cicaderma did not modify the level of GM-CSF
described to be essential for scarless wound repair. The low
level of GM-CSF is classically associated with a stronger
macrophage infiltration in the wound, with an increase of
angiogenesis and better healing (Fang et al., 2010). Thus, the
inability of Cicaderma to modify the GM-CSF level in this
model while down-regulating a few others (Fig. 7b) may be
hypothesized to contribute and promote faster wound healing
without keloid scar formation (Yeh et al., 2009). Indeed, the
lymphotactin level can be linked to the presence of PMNs,
which are the main producers of this cytokine. The reduction
of lymphotactin level induced by Cicaderma could be there-
fore correlated to the diminution of PMNs in the wound bed
and would be of particular interest for improving healing and
reducing the risk of keloid scar formation. In addition, the
slim, defined, and well organized collagen fibers in the Cica-
derma-treated group reinforce the improved quality of the
final remodeling of the wound, with less inflammation and no
keloid scar formation.
In conclusion, this study demonstrated that Cicaderma
ointment, a mix of well known natural extracts, modulates
inflammation, promotes re-epithelialization, and accelerates
skin wound healing in a skin ulcer model in mice (Fig. 9).
This ointment can be proposed to accelerate the healing of
small wounds, and its potential use in major diseases such as
burns and radiodermatitis should be considered. Moreover,
our data suggest the possibility it specifically regulates the
first steps of wound healing through the modulation of a few
cytokines, whose regulation clearly makes a potential target
for new pharmacological therapies in chronic wound pathol-
ogies. Finally, our data strengthen the emerging concept of a
sequential treatment to improve wound healing and involv-
ing different pharmacological tools.
Authorship Contributions
Participated in research design: Morin, Caredda, and Courty.
Conducted experiments: Morin, Roumegous, and Barbier-Chas-
sefie`re.
Performed data analysis: Morin, Roumegous, and Carpentier.
Wrote or contributed to the writing of the manuscript: Morin,
Garrigue-Antar, Caredda, and Courty.
Fig. 9. Sequence of events associated with normal and Cicaderma-treated wound healing.
Cicaderma’s Effect on Inflammation and Skin Wound Healing 123
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Address correspondence to: Morin Christophe, Laboratoire Croissance Cel-
lulaire, Re´paration, et Re´ge´ne´ration Tissulaires, E
´
quipe d’Accueil Convention-
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Cre´teil, 61 Avenue de Ge´ne´ral de Gaulle, 94010 Cre´teil, France. E-mail:
ch.morin@u-pec.fr
124 Morin et al.
