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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 remodeling that are associated with complex biochemical events. The interruption or failure of wound healing leads to chronic nonhealing wounds or fibrosis-associated diseases constituting 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 proinflammatory cytokines, mainly tumor necrosis factor (TNF)-α and others of the TNF pathway, which can be correlated to a decrease of polymorphonuclear recruitment. It is noteworthy that the modulation of inflammation through TNF-α, macrophage inflammatory protein-1α, interleukin (IL)-12, IL-4, and macrophage-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 treatments to improve healing.
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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
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.
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
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.
Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics 188599/3793692
JPET 343:115–124, 2012
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. 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 (
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.
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).
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. , 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. , 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. , 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. , 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-
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-
ne´e, Centre National de la Recherche Scientifique 7149, Universite´ Paris-Est
Cre´teil, 61 Avenue de Ge´ne´ral de Gaulle, 94010 Cre´teil, France. E-mail:
124 Morin et al.
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... Other diseases that complicate wound repair include hypotension, hypovolemia, edema, anemia, altered hormonal responses, nutritional deficiencies and poor hydration. Microbial infections would also delay the wound repair process and ultimately lead to chronic wound infection alongside recurrence issues [2,3]. Bacterial growth, which leads to wound bed colonization, could trigger a non-noticeable immune response that extends the inflammatory phase [4]. ...
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... Cicaderma® ointment (soft paraffin extract from Calendula officinalis L. Hyperycum. perforatum L., Achilea millefolium L., and mother tincture of Ledum palustre) (Laboratoire Boiron, Lyon, France) appeared as a promising treatment for burns and radiation-induced dermatitis [23]. Cicaderma® is indicated in the treatment of wounds, small superficial burns, and insect bites, and recommended to improve healing through inhibition of the inflammatory process and activity of matrix metalloproteinases [24][25][26]. ...
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... Since the mid-20th century, the Cicaderma ointment, prepared with Calendula officinalis L [Asteraceae], Hypericum perforatum L [Hypericaceae], and Achillea millefolium L [Asteraceae] extracts, has been marketed in Europe for the treatment of wounds, insect bites, and so on (Morin et al., 2012). Boiron Calendula cream/gel is another commercialized product in France for adjuvant treatment of irritant dermatitis and superficial burns (Pommier et al., 2004). ...
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Copaifera langsdorffii is a Brazilian native plant that is used for wound healing or as an anti-inflammatory agent. Non-healing wounds are an important health problem, particularly in horses, because they can cause the animal invalidity or even lead to death. In addition, horses respond to skin wounds with chronic inflammatory response and intense wound granulation, thereby delaying the healing process. By this way, our aim was to evaluate the healing potential of Copaifera langsdorffii hydroalcoholic extract (HE) and oil-resin creams (OR) in horse skin wounds. Four wounds were performed bilaterally in the lumbar region of six horses with a 2 cm punch and treated daily with the respective treatments: saline solution, vehicle, 10% HE creams or 10% OR cream. Daily planimetry analyses were performed to measure the wound area and clinical parameters. In four different experimental periods (3, 7, 14 and 21 d), wound biopsies were removed and used for microscopic analyses. SS wounds presented a significant small area at day 3 and 7, OR wounds presented significant small area in comparison with HE at 14 d, and no significant difference was observed between treatments at 21 d. A better microscopic and clinical healing activity of HE and OR was identified in comparison with the controls. The OR group showed better healing quality, specifically after 7 d of treatment. Therefore, Copaifera langsdorffii formulations demonstrated their wound healing potential in horse skin lesions, exhibiting an improvement of the macro- and microscopic parameters.
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Background: Skin wounds are closely correlated with opportunistic infections and sepsis risk. Due to the need of more efficient healing drugs, animal peptides are emerging as new molecular platforms to accelerate skin wound closure and to prevent and control bacterial infection. Aim: The aim of this study was to evaluate the preclinical evidence on the impact of animal peptides on skin wound healing. In addition, we carried out a critical analysis of the studies' methodological quality. Main Methods. This systematic review was performed according to the PRISMA guidelines, using a structured search on the PubMed-Medline, Scopus, and Web of Science platforms to retrieve studies published until August 25, 2020 at 3 : 00 pm. The studies included were limited to those that used animal models, investigated the effect of animal peptides with no association with other compounds on wound healing, and that were published in English. Bias analysis and methodological quality assessments were examined through the SYRCLE's RoB tool. Results: Thirty studies were identified using the PRISMA workflow. In general, animal peptides were effective in accelerating skin wound healing, especially by increasing cellular proliferation, neoangiogenesis, colagenogenesis, and reepithelialization. Considering standardized methodological quality indicators, we identified a marked heterogeneity in research protocols and a high risk of bias associated with limited characterization of the experimental designs. Conclusion: Animal peptides show a remarkable healing potential with biotechnological relevance for regenerative medicine. However, rigorous experimental approaches are still required to clearly delimit the mechanisms underlying the healing effects and the risk-benefit ratio attributed to peptide-based treatments.
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... As expected, GLSO decreased the IL-6, IL-1β and TNF-α levels of the wound. Interestingly, GLSO also reduced the levels of IL-4 and IL-10, which was reported to play an important role in delaying skin wound healing in their role as anti-inflammatory cytokines [36,37]. The reduction of pro-inflammatory and inflammatory cytokines showed the effect of GLSO on the regulation of inflammation. ...
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The mushroom Ganoderma lucidum (G. lucidum Leyss. ex Fr.) Karst has been a traditional Chinese medicine for millennia. In this study, we isolated the Ganoderma lucidum spore oil (GLSO) and evaluated the effect of GLSO on skin burn wound healing and the underlying mechanisms. Mice were used to perform skin wound healing assay. Wound analysis was performed by photography, hematoxylin/eosin staining, Masson's Trichrome staining and immunohistochemical analysis. Microbiota on the wounds were analyzed using the 16s rRNA sequence and quantitative statistics. The lipopolysaccharide (LPS) content was examined in skin wounds and serum using an enzyme-linked immunosorbent assay (ELISA). The expression of Toll-like receptor 4 (TLR4) and the relative levels of inflammatory cytokines were determined by qPCR and immunofluorescence assay. A pseudo-germfree mouse model treated with antibiotics was used to investigate whether GLSO accelerated skin burn wound healing through the skin microbiota. We found that GLSO significantly accelerated the process of skin wound healing and regulated the levels of gram-negative and gram-positive bacteria. Furthermore, GLSO reduced LPS and TLR4, and levels of some other related inflammatory cytokines. The assay with the pseudo-germfree mice model showed that GLSO had a significant acceleration on skin wound healing in comparison with antibiotic treatment. Thus, GLSO downregulated the inflammation by regulating skin microbiota to accelerate skin wound healing. These findings provide a scientific rationale for the potential therapeutic use of GLSO in skin burn injury.
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The inflammatory phase is an important event in the skin wound healing process. The deposition of granulation tissue in the wound bed and the rebuilding of the vascular network occur as inflammation diminishes. An angiogenic component in the formation of granulation tissue is the secretion of vascular endothelial growth factor, which assists in the chemotaxis, proliferation, and replication of fibroblasts. In this paper, we develop a mathematical model of skin wound healing angiogenic factors based on inflammatory cells (macrophages and neutrophils) and mediators (interleukin 6 and interleukin 10). We highlight the importance of this process in vascular endothelial growth factor release and in the formation of new capillary tips. We used a mathematical model of partial differential equations based on the reaction-diffusion-advection equations. In order to calibrate the parameters, we considered an in vivo model composed by four treatments: hydroalcoholic extract and oil-resin of Copaifera langsdorffii at 10% concentration, collagenase, and Lanette cream. Using the laboratory data for the wound edge, our mathematical model estimated the values of vascular endothelial growth factor concentration, and tips density in the center of the wound with a maximum error of 2.9%, and predicted healing time required for each treatment. The region of viability for the parameters, in the proposed model, was found through numerical simulations from the Interleukin 6 and 10 dysregulation and we obtained that, among the parameters analyzed, the greatest influencer in the dynamics of the system is the one, which represents the production of Interleukin 10 during phagocytosis.
Werner, Sabine, and Richard Grose. Regulation of Wound Healing by Growth Factors and Cytokines. Physiol Rev 83: 835–870, 2003; 10.1152/physrev.00032.2002.—Cutaneous wound healing is a complex process involving blood clotting, inflammation, new tissue formation, and finally tissue remodeling. It is well described at the histological level, but the genes that regulate skin repair have only partially been identified. Many experimental and clinical studies have demonstrated varied, but in most cases beneficial, effects of exogenous growth factors on the healing process. However, the roles played by endogenous growth factors have remained largely unclear. Initial approaches at addressing this question focused on the expression analysis of various growth factors, cytokines, and their receptors in different wound models, with first functional data being obtained by applying neutralizing antibodies to wounds. During the past few years, the availability of genetically modified mice has allowed elucidation of the function of various genes in the healing process, and these studies have shed light onto the role of growth factors, cytokines, and their downstream effectors in wound repair. This review summarizes the results of expression studies that have been performed in rodents, pigs, and humans to localize growth factors and their receptors in skin wounds. Most importantly, we also report on genetic studies addressing the functions of endogenous growth factors in the wound repair process.
We investigated the role of NO on expressional regulation of the chemotactic cytokine RANTES (regulated upon activation, normal T-cell expressed and secreted) during tissue regeneration using an excisional wound-healing model in mice. Wound repair was characterized by a large and sustained induction of RANTES expression, and inhibition of inducible nitric oxide synthase (iNOS) during repair only slightly decreased RANTES expression levels. Immunohistochemical analysis revealed keratinocytes of the wound margins and the hyperproliferative epithelium to be the main RANTES-expressing cell type within the wound. Therefore we analysed the regulation of RANTES expression in vitro in cultured human keratinocytes of the cell line HaCaT. Here we demonstrate that NO very efficiently suppressed interleukin-1β- and tumour-necrosis-factor-α-induced RANTES expression in keratinocytes. Furthermore, down-regulation of cytokine-induced RANTES mRNA in keratinocytes was dependent on endogenously produced NO, as inhibition of the co-induced iNOS by L-NG-monomethyl-L-arginine increased cytokine-triggered RANTES expression in the cells. Moreover, we observed strongest RANTES-immunopositive labelling in epithelial areas which were characterized by a NO-mediated low cellularity. Thus our data implicate NO as a negative regulator of RANTES expression during wound repair in vivo, as decreased numbers of keratinocytes observed in the absence of wound-derived NO might compensate for the high levels of RANTES expression which are associated with normal repair.
Conditioned medium from adipose-derived stem cells (ADSCs) stimulates both collagen synthesis and migration of dermal fibroblasts. However, it is still unknown whether conditioned media from tumor growth factor (TGF)-ss1-treated ADSCs (TGF-ss1-treated ADSCs-CM) induces increased expression of type I collagen, matrix metalloproteinase-1 (MMP-1), and migration as well as cell cycle regulatory proteins in fibroblasts, compared to non-treated ADSCs-CM. Our data showed that TGF-ss1-treated ADSCs-CM promoted effectively the proliferation and migration of human skin fibroblasts, compared to non-treated ADSCs-CM. In addition the expression of MMP-1 were markedly increased by treatment of TGF-ss1-treated ADSCs-CM in fibroblasts, compared to non-treated ADSCs-CM. Expression of type I collagen protein were slightly increased by treatment of TGF-ss1-treated ADSCs-CM in fibroblasts. The expression of cell cycle regulators of G1/S phase transition were not markedly altered by treatment of TGF-ss1-treated ADSCs-CM. Finally, artificial wounds were made using a 4-mm punch biopsy in hairless mice and TGF-ss1-treated ADSCs-CM were injected into the wound area. The injection of TGF-ss1-treated ADSCs-CM promoted the wound healing process in hairless mice. Taken together, our data indicated that TGF-ss1-treated ADSCs-CM induced up-regulation of type I collagen and MMP-1, promoted the migration of skin fibroblasts, and thereby promoted the wound healing process in vivo. Our data indicate that TGF-ss1-treated ADSCs-CM will be a component for a wound healing accelerating agent.
The development of a good blood supply is a key step in burn wound healing and appears to be regulated in part by myeloid cells. CX3CR1 positive cells have recently been identified as myeloid cells with a potential role in angiogenesis. The role of functional CX3CR1 system in burn wound healing is not previously investigated. A 2% contact burn was induced in CX3CR1(+/gfp) and CX3CR1(gfp/gfp) mice. These transgenic mice facilitate the tracking of CX3CR1 cells (CX3CR1(+/gfp)) and allow evaluation of the consequence of CX3CR1 functional knockout (CX3CR1(gfp/gfp)) on burn wound healing. The progression of wound healing was monitored before tissue was harvested and analyzed at day 6 and day 12 for migration of CX3CR1 cells into burn wound. Deficiency of a functional CX3CR1 system resulted in decreased recruitment of CX3CR1 positive cells into the burn wound associated with decreased myeloid cell recruitment (p<0.001) and reduced maintenance of new vessels (p<0.001). Burn wound healing was prolonged (p<0.05). Our study is the first to establish a role for CX3CR1 in burn wound healing which is associated with sub-dermal angiogenesis. This chemokine receptor pathway may be attractive for therapeutic manipulation as it could increase sub dermal angiogenesis and thereby improve time to healing.
Mechanical stretch has been shown to induce vascular remodeling and increase vessel density, but the pathophysiologic mechanisms and the morphologic changes induced by tensile forces to dermal vessels are poorly understood. A custom computer-controlled stretch device was designed and applied to the backs of C57BL/6 mice (n=38). Dermal and vascular remodeling was studied over a 7-day period. Corrosion casting and three-dimensional scanning electron microscopy and CD31 staining were performed to analyze microvessel morphology. Hypoxia was assessed by immunohistochemistry. Western blot analysis of vascular endothelial growth factor (VEGF) and mRNA expression of VEGF receptors was performed. Skin stretching was associated with increased angiogenesis as demonstrated by CD31 staining and vessel corrosion casting where intervascular distance and vessel diameter were decreased (p<0.01). Immediately after stretching, VEGF dimers were increased. Messenger RNA expression of VEGF receptor 1, VEGF receptor 2, neuropilin 1, and neuropilin 2 was increased starting as early as 2 hours after stretching. Highly proliferating epidermal cells induced epidermal hypoxia starting at day 3 (p<0.01). Identification of significant hypoxic cells occurred after identification of neovessels, suggesting an alternative mechanism. Increased expression of angiogenic receptors and stabilization of VEGF dimers may be involved in a mechanotransductive, prehypoxic induction of neovascularization.
To evaluate the efficacy of cultured cutaneous substitute (CCS) in accelerating the healing of combined radiation-skin wound injury (CRWI) in minipigs. Autologous porcine bone marrow-derived mesenchymal stem cells (BMSC) and skin-derived keratinocytes (SK) were infected by recombinant retrovirus expressing human (h) platelet-derived growth factor-A (hPDGF-A). CCS was constructed by loading acellular human amniotic membrane (HAM) with normal porcine BMSC and SK (BMSC-/SK-CCS) or with hPDGF-A modified counterparts (BMSC+/SK+CCS). The expression of exogenous hPDGF-A in cells and CCS was assessed by reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). The CCS or HAM were grafted to the dorsal CRWI sites (20 Gy local irradiation plus full-thickness skin removal, diameter = 40 mm) of minipigs. Wound healing rate and pathological changes were observed. High levels of hPDGF-A expression were confirmed in gene-modified cells (3780 pg/ml), cultured CCS (506 pg/ml) and transplanted CCS (250 pg/ml). The transplantation of the BMSC+/SK+CCS resulted in a shorter healing time (16-18, days) (P < 0.05 vs. other groups). The healing rates ranked as BMSC+/SK+CCS > BMSC-/SK-CCS > HAM > wound control. Pathologically, there were better granulation formation and re-epithelialisation, and collagen deposition in BMSC+/SK+CCS-treated wound than those in other groups. The angiogenesis ability followed the same order as healing rate of different groups. At day 7, the area densities of vasculature in granulation tissue of group BMSC+/SK+CCS, BMSC-/SK-CCS, HAM, wound only were 15.4, 10.3, 6.0 and 5.7%, respectively, while the number densities of vasculature was 767, 691, 126 and 109 (number/mm(2)), respectively. Topical transplantation of hPDGF-A modified CCS may be applicable to the management of refractory wounds.
After recruitment to the wound bed, monocytes differentiate into macrophages. Macrophages play a central role in all stages of wound healing and orchestrate the wound healing process. Their functional phenotype is dependent on the wound microenvironment, which changes during healing, hereby altering macrophage phenotype. During the early and short inflammatory phase macrophages exert pro-inflammatory functions like antigen-presenting, phagocytosis and the production of inflammatory cytokines and growth factors that facilitate the wound healing process. As such, the phenotype of wound macrophages in this phase is probably the classically activated or the so-called M1 phenotype. During the proliferative phase, macrophages stimulate proliferation of connective, endothelial and epithelial tissue directly and indirectly. Especially fibroblasts, keratinocytes and endothelial cells are stimulated by macrophages during this phase to induce and complete ECM formation, reepithelialization and neovascularization. Subsequently, macrophages can change the composition of the ECM both during angiogenesis and in the remodelling phase by release of degrading enzymes and by synthesizing ECM molecules. This suggests an important role for alternatively activated macrophages in this phase of wound healing. Pathological functioning of macrophages in the wound healing process can result in derailed wound healing, like the formation of ulcers, chronic wounds, hypertrophic scars and keloids. However, the exact role of macrophages in these processes is still incompletely understood. For treating wound repair disorders more should be elucidated on the role of macrophages in these conditions, especially their functional phenotype, to find more therapeutic opportunities. This review summarizes macrophage function in skin injury repair, thereby providing more insight in macrophage function in wound healing and possible interventions in this process.
While dermal substitutes can mitigate scarring and wound contraction, a significant drawback of current dermal replacement technologies is the apparent delay in vascular ingrowth compared with conventional skin grafts. Herein, we examined the effect of the chemokine stromal cell-derived factor-1 (SDF-1) on the performance of a porous collagen-glycosaminoglycan dermal analog in excisional wounds in mice. C57BL/6 mice with 1 cm × 1 cm dorsal full-thickness wounds were covered with a collagen-glycosaminoglycan scaffold, followed by four daily topical applications of 1 μg SDF-1 or phosphate-buffered saline vehicle. Some animals were also pretreated with five daily doses of 300 mg/kg granulocyte colony-stimulating factor. Animals treated with SDF-1 and no granulocyte colony-stimulating factor reepithelialized 36% faster than vehicle controls (16 vs. 25 days), and exhibited less wound contraction on postwounding day 18 (∼ 35% greater wound area) plus three-fold longer neoepidermis formed than controls. Conversely, granulocyte colony-stimulating factor promoted contraction and no epidermal regeneration. Early (postwounding Day 3) inflammatory cell infiltration in the SDF-1-treated group was 86% less, while the fraction of proliferating cells (positive Ki67 staining) was 32% more, when compared with controls. These results suggest that SDF-1 simultaneously delays contraction and promotes reepithelialization and may improve the wound-healing performance of skin substitutes.