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Rejuvenation Research: http://mc.manuscriptcentral.com/rejuvenationresearch
Role of trauma cytokines and erythropoietin
and their therapeutic potential for acute and chronicwounds
Journal: Rejuvenation Research
Manuscript ID: REJ-2010-1050.R1
Manuscript Type: Clinical Articles
Date Submitted by theAuthor:
02-Jul-2010
Complete List of Authors: Bader, Augustinus; Biotechnological Biomedical Center, Departmentof Cell Techniques and Stem Cell Biology, University of LeipzigLorenz, Katrin; Biotechnological Biomedical Center, Department ofCell Techniques and Stem Cell Biology, University of Leipzig
Richter, Anja; Biotechnological Biomedical Center, Department ofCell Techniques and Stem Cell Biology, University of LeipzigScheffler, Katja; Biotechnological Biomedical Center, Department ofCell Techniques and Stem Cell Biology, University of LeipzigKern, Larissa; Biotechnological Biomedical Center, Department ofCell Techniques and Stem Cell Biology, University of LeipzigEbert, Sabine; Biotechnological Biomedical Center, Department ofCell Techniques and Stem Cell Biology, University of LeipzigGiri, shibashish; Biotechnological Biomedical Center, Department ofCell Techniques and Stem Cell Biology, University of LeipzigBehrens, Maria; Medical Writing ExpertsDornseifer, Ulf; Klinikum Bogenhausen, Department of Plastic,Reconstructive, Hand and Burn SurgeryMacchiarini, Paolo; Hospital Clinico de Barcelona, Barcelona,4Department of General Thoracic Surgery
Machens, Hans-Gunther; Klinikum Rechts der Isar, TechnischeUniversitt Mnchen, 5Department of Plastic and Hand Surgery
Keyword:Regeneration, Skin Aging, Stem Cells, Quality of Life, GrowthFactors
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
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Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
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The interactive role of trauma cytokines and erythropoietin
and their therapeutic potential for acute and chronic wounds
Augustinus Bader1, Katrin Lorenz
1, Anja Richter
1, Katja Scheffler
1, Larissa Kern
1, Sabine
Ebert1, Shibashish Giri
1, Maria Behrens
2, Ulf Dornseifer
3, Paolo Macchiarini
4, Hans-Gnther
Machens5
1University of Leipzig, Centre for Biotechnology and Biomedicine, Department of Applied
Stem Cell Biology and Cell Techniques, Germany
2Medical Writing Experts, Langwedel, Germany
3Klinikum Bogenhausen, Zentrum fr Schwerbrandverletzte, Mnchen
4Hospital Clinico de Barcelona, Dept. of General Thoracic Surgery
5Klinik fr Plastische Chirurgie, Klinikum Rechts der Isar, Technische Universitt Mnchen,
Germany
Abstract:
If controllable, stem cell activation following injury has the therapeutic potential for
supporting regeneration in acute or chronic wounds. Human dermally derived stem cells
(FmSCs) were exposed to the cytokines IL-6, IL-1 and TNF- in the presence of
erythropoietin. Cells were cultured under ischemic conditions and phenotypically
characterized using flow cytometry. Topical EPO application was performed in three
independent clinical wound healing attemps. The FmSCs expressed the receptor for
erythropoietin (EPO). EPO had a strong inhibitory effect on FmSC growth in the absence of
IL-6 and TNF-. With IL-6, the EPO effects were reversed to that of growth stimulation.
TNF- had the strongest stimulatory effect. In contrast, IL-1 had an inhibitory effect.
Topically applied EPO considerably enhanced wound healing and improved wound
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conditions of acute and chronic wounds. Site specificity of stem cell activation is mediated by
IL-6 and TNF-. In trauma, EPO ceases its inhibitory role and reverts to a clinically relevant
boosting function. EPO may be an important therapeutic tool for the topical treatment of acute
and chronic wounds.
INTRODUCTION
The human body has command of a tool box that allows it to appropriately react to injury with
an amazing site specificity to achieve a regenerative response. It has been unclear how local
stem cells are awakened in such instances of need. If this mechanism was better understood,
a fundamental therapeutic strategy that would allow site-specific stem cell activation in any
area of the human body could be developed. Site specificity of tissue regeneration has been
taken for granted as a normal process, but their underlying mechanisms, relevance for stem
cell-based responses and therapeutic potential have not yet been understood. Frequently, this
innate capacity is not sufficient to lead to full restoration, resulting in so-called nonhealing
wounds.
It is well-known that local trauma leads to the release of inflammatory cytokines, including
IL-6, IL-1 and TNF-.1
Mesenchymal stem cells (MSCs), isolated from many human tissues,
such as bone marrow, adipose tissue, the adult liver, peripheral blood, amniotic fluid, the
bronchial lung, the articular synovium and other fetal tissues, are a cell population that
possesses a fibroblastic-like morphology, limited but long-term viability, self-renewal
capacity and multilineage potential.2,3
They are characterized by similar surface antigen
expression patterns for CD14(-), CD31(-), CD34(-), CD45(-), CD71(+), CD73/SH3-SH4(+),
CD90/Thy-1(+), CD105/SH2(+), CD133(-) and CD166/ALCAM(+)4,5
. MSCs can
differentiate into adipogenic, osteogenic, myogenic, chondrogenic and neurogenic cell types.
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In an effort to identify new sources of mesenchymal stem cells, dermal rodent fibroblast cell
lines were examined for their mesenchymal potential.9-11
Publications by Toma et al.9
and
Crigler et al.10
suggest that the adult mammalian dermis contains tissue-derived stem cells
and that these fibroblastic mesenchymal stem cells are more plastic than previously
appreciated. Dermis-derived fibroblastic mesenchymal stem cells have been used for
therapeutic applications such as transplantation to support bone formation.12-14
Toma et al.9
demonstrated the mesenchymal plasticity of primary human dermal fibroblasts in vitro with
different approaches regarding characterization and applications.15-18
Zuk et al. analyzed the
phenotypic characteristics of these fibroblasts and noted that their phenotype seems to be
similar to that of adult-derived stem cells (ADSCs).5,16,18
To continue these studies, we examined whether unselected human dermis fibroblastic
mesenchymal cells possess stem cell-like characteristics and are phenotypically similar to
ADSCs. Bone marrow-derived MSCs (bmMSCs) have been shown to support the wound
healing of chronic skin wounds. Badiavas and Falanga (2003) demonstrated that chronic skin
ulcers of patients with arterial and venous insufficiency were healed with complete wound
closure and less scar formation when treated with bmMSC-seeded grafts.21
To characterize FmSCs relative to ADSCs, we analyzed the cytoskeletons and compositions
of the extracellular matrix as well as the mesenchymal phenotypes and differentiation
properties of both. The obtained data show for the first time that primary human dermal
fibroblasts in vitro share common characteristics with ADSCs, such as phenotype and
differentiation potential. Our results demonstrated that FmSCs fulfill the three main
characteristics of MSCs: they express all MSC-related surface antigens homogenously; their
cytoskeleton and matrix compositions are quite similar to that of MSCs; and they differentiate
along the adipogenic and osteogenic cell lineages20
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Both conditions, however, do not sufficiently explain the mechanism of endogenous stem cell
activation in the case of injury alone.
We therefore developed an in vitro model of trauma conditions by investigating the role of IL-
6, IL-1 and TNF- on human skin stem cells, identified a receptor for Erythropoietin (EPO)
in these cells and investigated the role of EPO with and without the presence of the trauma
cytokines. The knowledge obtained from these studies was then transferred to three
independent and specific clinical cases: EPO was topically applied to a split-thickness skin
graft donor site, a pressure and a vascular ulcer.
EPO has been used in clinical practice for a wide range of diseases22
, obtaining systemic
application via subcutaneous, intramuscular or intravenous route.23-28
A topical administration
to stem cells at the site of the wound injury would allow a more direct stimulatory response
and would work only if an appropriate interference with site-specific mechanistic responses
occurred. The elucidation of such mechanisms and the development of a therapeutic potential
has been the scope and success of this study.
METHODS
Cell isolation
Human juvenile foreskin samples were obtained from four-year-old patients undergoing
circumcision after written consent was obtained. This study was approved by the ethics
commission of Leipzig University and was conducted in accordance with the Declaration of
Helsinki protocols.
Epidermal and dermal tissue were isolated by mechanical and enzymatic digestion, as
previously described by Ponec et al29
After removing the epidermis from the dermis the
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tissue was cut into small pieces and washed three times with sterile phosphate-buffered saline
(PBS) at room temperature. The pieces were then incubated with 0.075% collagenase type A
(Roche Diagnostics, Mannheim, Germany) for 12 h at 37C with gentle agitation.
For FmSC suspensions, the enzymatic reaction was inactivated with DMEM/10% FBS
(Gibco/Invitrogen, Karlsruhe, Germany) and filtered through a 70-m mesh. This cell
suspension was centrifuged at 600 x g for 5 min. The cell pellet was then gently resuspended
in DMEM/10% FBS, filtered through a 70-m mesh and plated in conventional T75 tissue
culture flasks (BD Falcon, Heidelberg, Germany). Cells were cultured in DMEM
supplemented with 10% FBS, GlutaMAX-I, 4.5 g/L glucose and pyruvate (Gibco/Invitrogen).
Cell proliferation
FmSCs were seeded in six-well plates at passage 4. After one day of cultivation in
DMEM/10% FBS, the cells attached and adapted to start proliferation. On the following day,
the cells were cultured with the same medium but without FBS to minimize serum-induced
effects. The next day, cytokine stimulation of the cells was initiated with cultivation in
DMEM/10% FBS supplemented with or without 10 ng/mL EPO in combination with 10
ng/mL IL-6, 10 ng/mL IL-1 or 10 ng/mL TNF-. Controls were also performed with each
cytokine alone. At days 3, 5, 7 and 11, cells were trypsinized and viable cell numbers were
counted in a hemocytometer by trypan blue staining. All experiments were done in triplicate,
with three independent sets of patient materials.
Cell immunophenotyping
Immunophenotyping of FmSCs was performed as described previously by Lorenz et al19
. The
following labeling reagents were used: fluorescein isothiocyanate (FITC)- or phycoerythrin
(PE)-conjugated mouse antibodies, anti-human CD31 (Biozol Diagnostica, Munich,
Germany) anti-human CD45 (Sigma-Aldrich Seelze Germany) anti-human CD90 and anti-
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human CD105 (BD Biosciences, Heidelberg, Germany) and anti-human CD166 (Acris
Antibodies, Hiddenhausen, Germany). The monoclonal antibody mouse anti-human CD73
(BD Biosience) was unlabeled and combined with a secondary PE-labeled goat anti-mouse
antibody (Sigma-Aldrich). Incubation and flow cytometry analyses were performed according
to conventional techniques29
. Isotype controls were equally concentrated, labeled or
unlabeled. The stained cells were analyzed on a FACS Calibur (BD Bioscience) using
CellQuest Pro (BD Bioscience). Fluorescence intensities were determined by flow cytometry
in a minimum of 1x104
cells.
Growth curves
To record a growth curve, three individual tests were performed, each in triplicate. Cell
counting was performed at day 0, 1, 3, 5 and 7 by trypsinization and trypan blue staining
using a Neubauers chamber. The cells were seeded at passage 6 with a density of 20,000
cells/well (9.6 cm2) in a six-well plate (Falcon) two days before stimulation (day 0). The
exchange of media one day before stimulation from 10% FBS-containing DMEM to serum-
free DMEM ensured the attachment of the cells during the last 24 hours and a minimal protein
background from the FBS. The stimulation was made with or without the presence of IL-6,
IL-1 and TNF- by adding EPO (NeoRecormon, Roche) and/or the cytokines (all
RELIAtech) to the media and performing a full media exchange. At days 3 and 5, half the
volume of the media was changed with media containing the initial concentrations of EPO
and/or cytokine. The cell scores of each sample and the average of all nine samples were
calculated, including the standard deviation. To compare the different growth curves, a
Students t-test was used to test the significance of the differences between the breakpoints.
For stimulation with cytokines, a concentration-dependent pretest was performed to identify
the minimum concentration needed for successful stimulation; this was determined to be 10
ng/mL
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Clinical cases
In three highly different clinical situations, topical EPO application was used to support
wound healing. All patients provided informed consent based on the guidelines of the local
ethical committee and the national legal requirements in Germany. Topical treatment was
performed using a mixture of 3,000 IE erythropoietin- (NeoRecormon, F. Hoffmann-La
Roche AG, Basel, Schweiz) and 20 g hydrogel (Varihesive, ConvaTec, NJ, USA). In patient
A, the mixture was topically applied to a 0.3-mm deep split-thickness skin graft donor sites
measuring 8 x 24 cm directly after skin harvesting. The donor site at the thigh was
subsequently closed with a polyurethane dressing (OpSite, Smith&Nephew, London, UK).
After three and six days, the mixture was again applied by puncturing the polyurethane film,
which remained on the wound. At day 7, the film dressing was removed to evaluate the
reepithelialization. Another donor site of equal depth and dimension at the contralateral leg of
patient A was treated similar but the mixture was replaced by hydrogel alone - without EPO.
Again the dressing rest in place for seven days followed by dressing removal and wound
assessment. The standardized wound management provided an ideal comparability of both
similar wounds.
Patient B had a non-healing pressure sore at the heel following urosepsis and Patient C had a
non-healing vascular ulcer. In both patients the wounds were surgically debrided and treated
in the same manner, using the same mixture of EPO and hydrogel. Moist wound management
was provided by covering both wounds with Varihesive (Convatec, Skillman, NJ, USA). A
total of five dressing changes with new EPO applications were performed in both patients to
prepare the wound for skin grafting. The poor general condition of patient B and C did not
allow extensive reconstructional procedures. Therefore, the aim of local EPO application was
to prepare the wound bed in a manner that enables subsequent successful skin grafting. And to
avoid lower leg amputation by a minor surgical procedure
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RESULTS
Sequencing the EPO receptor in human FmSCs
Cells were characterized for their expression of CD31, CD45, CD73, CD90, CD105 and
CD166. The expression of CD34, CD71 and CD133 was also examined. Fig. 1a shows the
mRNA expression profile of the EPO receptor in FmSCs. Sequencing of the PCR product for
the mRNA of the EPO receptor showed 90-98% sequence homology.
Figure 1
Figure 2
Figure 3
Switch from inhibitory to stimulatory effects of EPO on stem cell proliferation
Cells were cultured from human biopsies and grown to the 4th passage in vitro. Stimulation of
EPO alone in the absence of any cytokines showed an inhibitory effect on stem cell growth
(Fig. 1b). Cells under control conditions grew up to 1.545 million cells. In contrast, we
observed a dramatic decrease in cell proliferation when the FmSCs were stimulated with
EPO. Cell proliferation was minimized by 32%, to a total cell number of 1.053 million cells.
IL-6 stimulation of FmSCs also resulted in decreased growth activity relative to the control
cells, but this was reversed in the presence of EPO (Fig. 1c). This indicates both an inhibitory
role of EPO (Fig. 1b) in the absence of cytokines or in the late phase of trauma and a
supportive, boosting activity of EPO in the presence of IL-6 (Fig. 1c). TNF- was a strong
stimulator of FmSC proliferation, and the presence of EPO did not influence this. Cell
proliferation was elevated most with TNF-. IL-1 had an inhibitory effect on the
proliferation of the stem cells compared to controls cells, both with and without EPO.
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Effect of stimulation on the expression of stem cell markers
To characterize the FmSCs, surface antigens were analyzed by flow cytometry. FmSCs
homogenously expressed CD90, CD73, CD105 and CD166. (Fig. 2, Fig. 3) In contrast,
expression of endothelial cell surface markers such as CD31 was not detected. In addition,
hematopoietic cell subpopulations positive for surface antigens such as CD45, CD14 and
CD133 were not observed.
During cultivation and stimulation of FmSCs with inflammatory cytokines in combination
with EPO, we did not detect any changes in the surface antigen expression of MSC markers.
Except when cultivating FmSCs with IL-6 in combination with EPO, we found changes in the
expression of CD90. This suggests that the unselected stem cell population was stimulated
differently, and thus two different CD90-expressing cell populations were detected. (Fig. 3)
In vivo experiments
The more complex in vivo situation is characterized by an intricate interplay of cytokine
profiles, consisting of mixtures and time-sequence variations with respect to availability.
In Patient A, complete and stable reepithelialization of the split-thickness skin graft donor
site, topically treated with EPO, was achieved seven days after the operation. The wound
surface was closed, dry and clearly looked pale. (Fig.4a) In contrast, the donor site at the
contralateral thigh that was treated without EPO showed incomplete reepithelialization at this
time point, indicated by secretion and a dark-red wound surface. (Fig.4a)
In Patient B, sufficient granulation tissue formation was obtained after five local treatment
sessions with EPO, which provided an highly vascularized wound bed for sucessful split-
thickness skin grafting. The ideal prepared wound bed enabled salvage of the limb without
extensive reconstructive procedures (Fig 4b)
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In Patient C, improved healing and sufficient granulation tissue formation was achieved
following five local applications of EPO. (Fig.4c) After subsequent skin grafting at days 21
and 56, the wound healed well and has remained stable for more than 12 months (Fig. 4c).
DISCUSSION
Erythropoietin is a type I cytokine that was approved by the US Food and Drug
Administration (FDA) in 1989 for the treatment of the anemia of end-stage renal disease.
Thereafter, erythropoietin has been used for the treatment of a diverse range of diseases,
including cancer28,31,32
, heart care erythropoiesis33-37
, malaria38
, ischemic and degenerative
damage of neurons40
, retinopathy40,41
and diabetic retinopathy42-45
. EPO plays a crucial role in
the process of endochondral ossification in bone repair in mice via EPO-receptor expression46
.
Gough (2008)47
supported the concept that understanding the EPO receptors by which EPO
signaling contributes to organ development provides information on the differentiation of
erythrocytes. Interestingly, Foster et al. (2004)48
found increased EPO-R protein levels in
dynamically growing canine lungs after pneumonectomy, suggesting a paracrine role for EPO
signaling in lung growth and remodeling. This hypothesis may be applicable to other types of
organ repair since EPO and EPO-R are expressed in several organs (e.g., kidney, brain, heart,
muscle and endothelial cells)49
. However, it is now known that EPO and EPO-R are local
products in a wide range of cells that specifically protect other cells from potentially cytotoxic
events and metabolic stress.
Adding to the evidence of Bodo et al. (2007)50
that normal human skin expresses EPO and
functional EPO-R, our study showed that skin stem cells specifically are the responsive
elements in normal skin containing the receptor for EPO and thus show a special readiness for
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action in the case of traumatic skin injuries. In the case of injury and ischemic trauma,
cytokines IL-6, IL-1 and TNF- are alerted. We demonstrated that the trauma cytokine IL-6
and EPO synergistically up-regulated stem cell growth in the case of hypoxic skin conditions.
In contrast, without trauma, EPO exerted an inhibitory effect on in vitro skin stem cells. This
effect reverted to stimulation in the presence of IL-6 and TNF- . Only IL-1 maintained its
inhibitory function with or without EPO. Neither the trauma cytokines nor EPO grossly
changed the phenotype of the fibroblast precursor cells.
Our findings agree with the observations of Paus et al. (2009)51
that the oxygen sensing skin
response is mediated by skin EPO. In situations of low oxygen, skin EPO and IL-6 are
alarmed to react to the site-specific injury. This finding compliments the relevance of our data
as a physiological and potentially highly relevant therapeutic strategy for endogenous stem
cell activation in the case of trauma.
In the human scalp, it was shown that hair follicles expressed EPO at the mRNA and protein
levels, up-regulated EPO transcription under hypoxic conditions and expressed transcripts of
EPO-R and the EPO stimulatory transcriptional cofactor hypoxia-inducible factor-1. These
findings are in line with recent research results that showed that hair follicle-derived
keratinocytes were a major cell source for reepithelialization during wound healing52
and that
the hair follicle connective tissue sheath was a source of granulation tissue formation53
.
Boutin et al. (2008)54
revealed the EPO-connected oxygen sensing functions of the skin and
elucidated how mammalian cells adapt to low oxygen levels by recruiting the skin as a central
coordinator of the systemic response to hypoxia. Hair follicles are able to detect insufficient
oxygen levels, a crucial mechanism of the extremely fast renewing and proliferating cell
population, to regulate its metabolic balance. Using transgenic mice studies, Kochling (1998)
revealed that the hypoxia response elements are located upstream (between 9.5 and 14 kb) of
the EPO gene in the kidney and downstream (within 0 7 kb) of the EPO gene in the liver It
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has been shown that the circulating levels of EPO may increase up to 1,000-fold in response
to hypoxia in the kidney53
.
In the absence of trauma cytokines, EPO down-regulates the proliferation of skin stem cells in
vitro. In the case of traumatic skin hypoxia, IL-6 and TNF- activate stem cells. Specifically,
in the presence of IL-6, the inhibitory role of EPO is reversed to increase stem cell
proliferation. This represents an adequate response to a pathophysiological need. The
stimulatory effects of TNF- are not diminished by the previously inhibitory role of EPO.
Among the trauma cytokines studied here, only IL-1 also exhibited an inhibitory function
that did not interfere with the original inhibitory role of EPO. In vivo, we observed the net
effect of cytokine and EPO stimulation in acute and chronic wound types. In both cases, the
regenerative response was boosted qualitatively and quantitatively. By these mechanisms, the
skin trauma EPO system switches from its inhibitory function to a supportive role for
boosting skin regeneration. The inflammatory activity of the wound itself represents a
permissive situation for the boosting activity of EPO, which seems to reduce the healing time
at split-skin graft donor sites from 10 to 7 days. In the previously non-healing wounds, EPO
assumes an enabling role that shifts the balance from non-healing to healing and triggers the
formation of granulation tissue. The expression of EPO-R on the stem cells suggests that this
could be a normal role of EPO that permits the human body to recover from site-specific
tissue damage or injury in any area of the human body. This mode of action has been
demonstrated clinically by the clearly accelerated reepithelialization of the EPO treated donor
site in direct comparison to the non-EPO treatet donor site at the same patient.
Non-healing chronic wounds are at the opposite end of the spectrum of acute wound-healing
mechanisms, progressing toward healing at a different rate. In the case of diabetic patients,
this represents a critical situation for surgical practice as approximately 22 million diabetic
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patients suffer from chronic wounds57,58
, with many of them suffering from non-healing
chronic wounds59
. There are approximately 5.2 million pressure ulcers and 7.6 million venous
ulcers in the world that require treatment every year.
Fibroblasts form granulation tissue via hyper-proliferation. This leads to a normal process of
not only cellular rebuilding of lost dermal tissue but also reconstitution of the physical barrier
of the basal lamina and the scaffold for revascularization. Large-area wounds do not heal
within a short time, so the risk of infection and dehydration rises dramatically. Our results
suggest that we can completely alter the wound-healing landscape and have a major impact on
the care of both acute and chronic wounds. This study provides mechanistic evidence to
support the hypothesis that this novel treatment modality physically modifies the wound
microenvironment and thereby promotes wound healing in clinical relevant manner.
In a few clinical applications, fibroblasts were used to treat diabetic or venous ulcers60-62
, but
this methodology remains controversial. We report a mechanism explaining how endogenous
stem cells can be activated locally at the site of a severe wound without necessitating the
transplantation of exogenous cells. All clinical cases, although diverse in their
pathophysiology, were dramatic successes with respect to their respective healing responses.
Brines & Cerami described63
that EPO is locally produced in the immediate surrounding area
of a tissue injury to counteract the destructive effects of cytokines such as TNF- by
preventing cell apoptosis, thus the development of secondary, proinflammatory cytokine-
induced injury can be reduced. However, a delicate balance in tissue injury exists between
EPO and proinflammatory cytokines such as TNF-. Therefore, compensatory EPO
production by nearby tissue balances the effects of inflammatory mediators and prevents the
further spread of damage63
Hamed et al reported that treatment with topical EPO improves
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the defect repair of excised wounds in diabetic rats.64
They suggested that vascular VEGF-
induced angiogenesis, enhanced collagen deposition and reduced apoptosis in the diabetic
wound bed are among the mechanisms that underlie the effects of topical EPO. This work by
Hamed et al.64
was the first to investigate the use of topical EPO treatment for wound healing.
However we are the first of topical EPO treatment for acute and chronic wounds patients.
Although other cytokines such as tumor growth factor-, monocyte chemoattractant protein-1
and colony-stimulating factor-1 are released from the invading inflammatory cells to the
wound bed upon skin injury and in chronic wounds65
, we selected these cytokines (IL-6 ,
TNF- and IL-1 ) because these are leading cytokines which is associated with organ
trauma injury including chronic wound.66
Despite the beneficial effect on wound repair, one has to assume that doses of EPO are rather
high within the defect wound and low systemically. Rezaeian et al67
demonstrated that a
triplicate intraperitoneal dose of 500IU EPO/kg bw over 48 hours did not influence RBC
count and Hematocrit, whereas Galeano et al68
observed a significant increase in RBC count
and hemoglobin after 12 days of daily subcutaneous administration of 400IU EPO / kg bw. In
compared to this, the EPO concentration of our present clinical study is 50 U (one time) by
topical application of the hydrogel containing EPO in the patients. This concentration (50U) is
75 times less than other existing dose of various other experimental or clinical models. Using
this concentration, we are conducting multicenter clinical trials for actute and choric wound
pateints. Everytime fresh hydrogel is prepared and half life of EPO is 48 hours and stable in
gel up to 12 weeks. There is no systemic effect of treated patients which is main advantages
of this topical application. We measured red blood cell (RBC) count and haemoglobin,
leukocyte and platelet count of the patients before and after EPO treatment but there were no
any difference.
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Our present investigation may provide a standard supplemental therapy for reducing the
mortality and morbidity associated with chronic wounds, especially in the elderly, the
disabled and those with diabetes. Especially large-area burn injuries, where the wound closure
is a race against time, may benefit from the healing accelerating characteristics of EPO. Not
only the burned, debrided and grafted areas but also the skin graft donor sites have to heal in a
limited time frame. Frequently, donor sites do not rejuvenate for reharvesting as fast as
needed, resulting in graft deficiencies that may lead to further extensive complications with
fatal outcomes. The targeted clinical areas will improve in the assistance toward accelerating
regeneration of acute and chronic wounds, and endogenous stem cell activation may reduce
the need for skin grafting of burns.
References
1. Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med 1999;341:738-46.
2. Mller T, Ander L, Kolf K, Woitalla D, Muhlack S. Comparison of 200 mg retarded
release levodopa/carbidopa - with 150 mg levodopa/carbidopa/entacapone application:
pharmacokinetics and efficacy in patients with Parkinson's disease. J Neural Transm
2007;114:1457-1462.
3. Bobis S, Jarocha D, Majka M. Mesenchymal stem cells: characteristics and clinical
applications. Folia Histochem Cytobiol 2006;44:215-230.
4. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA,
Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal
stem cells. Science 1999;284:143-147.
5. Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, Alfonso ZC, Fraser JK,
Benhaim P, Hedrick MH. Human adipose tissue is a source of multipotent stem cells. Mol
Biol Cell 2002;13:4279-4295
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6. Hauner H, Schmid P, Pfeiffer EF. Glucocorticoids and insulin promote the differentiation
of human adipocyte precursor cells into fat cells. J Clin Endocrinol Metab 1987;64:832-835.
7. Grigoriadis AE, Heersche JN, Aubin JE. Differentiation of muscle, fat, cartilage, and bone
from progenitor cells present in a bone-derived clonal cell population: effect of
dexamethasone. J Cell Biol 1988;106:2139-2151.
8. Wakitani S, Saito T, Caplan AI. Myogenic cells derived from rat bone marrow
mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve 1995;18: 1417-1426.
9. Toma JG, Akhavan M, Fernandes KJ, Barnabe-Heider F, Sadikot A, Kaplan DR, Miller
FD. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell
Biol 2001;3:778-784.
10. Crigler L, Kazhanie A, Yoon TJ, Zakhari J, Anders J, Taylor B, Virador VM. Isolation of
a mesenchymal cell population from murine dermis that contains progenitors of multiple cell
lineages. Faseb J 2007;21:2050-2063.
11. Toma JG, Akhavan M, Fernandes KJ, Barnabe-Heider F, Sadikot A, Kaplan DR, Miller
FD. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell
Biol 2001;3:778-784.
12. French MM, Rose S, Canseco J, Athanasiou KA. Chondrogenic differentiation of adult
dermal fibroblasts. Ann Biomed Eng 2004;32:50-56.
13. Hirata K, Tsukazaki T, Kadowaki A, Furukawa K, Shibata Y, Moriishi T, Okubo Y,
Bessho K, Komori T, Mizuno A, Yamaguchi A. Transplantation of skin fibroblasts expressing
BMP-2 promotes bone repair more effectively than those expressing Runx2. Bone
200;32:502-512.
14. Krebsbach PH, Gu K, Franceschi RT, Rutherford RB. Gene therapy-directed
osteogenesis: BMP-7-transduced human fibroblasts form bone in vivo. HumGene Ther.
2000;11:1201-1210
ge 17 of 29 Rejuvenation Research
8/7/2019 Rejuvenation Research_Role of Trauma_accepted
19/31
ForPeer
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15. Rutherford RB, Moalli M, Franseschi RT, Wang D, Gu K, Krebsbach PH. Bone
morphogenetic protein-transduced human fibroblasts convert to osteoblasts and form bone in
vivo. Tissue Eng 2002;8:441-452.
16. Lysy PA, Smets F, Sibille C, Najimi M, Sokal EM. Human skin fibroblasts:From
mesodermal to hepatocyte-like differentiation. Hepatology 2007;46:1574-1585.
17. Chen FG, Zhang WJ, Bi D, Liu W, Wei X, Chen FF, Zhu L, CuiL, Cao Y. Clonal analysis
of nestin(-) vimentin(+) multipotent fibroblasts isolated from human dermis. J Cell Sci
2007;120:2875-2883.
18. Toma, JG, McKenzie IA, Bagli D, Miller FD. Isolation and characterization of
multipotent skin-derived precursors from human skin. Stem Cells 2005;23:727-737.
19. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP,
Hedrick MH. Multilineage cells from human adipose tissue: implications for cell-based
therapies. Tissue Eng 2001;7:211-228.
20. Lorenz K, Sicker M, Schmelzer E, Rupf T, Salvetter J, Schulz-Siegmund M, Bader A.
Multilineage differentiation potential of human dermal skin-derived fibroblasts. Exp Dermatol
2008b;17:925-932.
21. Badiavas EV, Falanga V. Treatment of chronic wounds with bone marrowderived cells.
Arch Dermatol 2003;139:510-516.
22. Noguchi CT, Wang L, Rogers HM, Teng R, Jia Y. Survival and proliferative roles of
erythropoietin beyond the erythroid lineage. Expert Reviews in Molecular Medicine
2008;1:36.
23. Amarguellat F, Gogusev J, and Drueke TB. Direct effect of erythropoietin on rat vascular
smooth-muscle cell via a putative erythropoietin receptor. Nephrol Dial Transplant
1996;11:687-692.
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8/7/2019 Rejuvenation Research_Role of Trauma_accepted
20/31
ForPeer
Review
24. Alvarez Arroyo MV, Castilla MA, Gonzalez Pacheco FR, Tan D, Riesco A, Casado S,
Caramelo C. Role of vascular endothelial growth factor on erythropoietinrelated endothelial
cell proliferation. Journal of American Society on Nephrology 1998;9:1998-2004.
25. Buemi M, Marino D, Floccari F, Ruello A, Nostro L, Aloisi C, Marino MT, Di Pasquale
G, Corica F, Frisina N. Effect of interleukin 8 and ICAM-1 on calcium-dependent outflow of
K+ in erythrocytes from subjects with essential hypertension. Current Medical Research and
Opinion 2004;20:19-24.
26. Buemi M, Vaccaro M, Sturiale A, Galeano MR, Sansotta C, Cavallari V, Floccari F,
DAmico F, Torre V, Calapai G, Frisina N, Guarneri F, Vermiglio G. Recombinant human
erythropoietin influences revascularization and healing in a rat model of random ischaemic
flaps. Acta Derm Venereol 2002;82:411-417.
27. Vaccaro M, Magaudda L, Cutroneo G, Trimarchi F, Barbuzza O, Guarneri B. Changes in
the distribution of laminin a1 chain in psoriatic skin. Immunohistochemical study using
confocal laser scanning microscopy British Journal of Dermatology 2002;146:392-398.
28. Bennett CL, Silver SM, Djulbegovic B, Samaras AT, Blau CA, Gleanson KJ, Barnato SE,
Elvermann KM, Courtney DM, McKoy JM, Edwards BJ, Tigue CC, Raisch DW, Yarnold PR,
Dorr DA, Kuzel TM, Tallman MS, Trifilio SM, West DP, Lai SY, Henke M. Venous
thromboembolism and mortality associated with recombinant erythropoietin and darbepoetin
administration for the treatment of cancer-associated anemia. JAMA 2008;299:914-924.
29. Ponec M, Weerheim A, Kempenaar J, Mommaas AM, Nugteren DH. Lipid composition
of cultured human keratinocytes in relation to their differentiation. J Lipid Res 1988;29:949-
61.
30. Brouty-Boye D, Raux H, Azzarone B, Tamboise A, Tamboise E, Beranger S, Magnien V,
Pihan I, Zardi L, Israel L. Fetal myofibroblast-like cells isolatedfrom post-radiation fibrosis in
human breast cancer. Int J Cancer 1991;47:697-702.
ge 19 of 29 Rejuvenation Research
8/7/2019 Rejuvenation Research_Role of Trauma_accepted
21/31
ForPeer
Review
31. Jelkmann W, Bohlius J, Hallek M, Sytkowski AJ. The erythropoietin receptor in normal
and cancer tissues. Critical Review on Oncology and Hematology 2008;67:39-61.
32. Littlewood TJ, Bajetta E, Nortier JW, Vercammen E, Rapoport B, Epoietin Alpha Study
Group. Effects of epoetin alfa on hematologic parameters and quality of life in cancer patients
receiving nonplatinum chemotherapy: results of a randomized, double-blind, placebo-
controlled trial. Journal of Clinical Oncology 2001;19:2865-2874.
33. Burger D, Lei M, Geoghegan-Morphet N, Lu X, Xenocostas A, Feng Q. Erythropoietin
protects cardiomyocytes from apoptosis via up-regulation of endothelial nitric oxide synthase.
Cardiovascular Research 2006;72:51-59.
34. Calvillo L, Latini R, Kajstura J, Leri A, Anversa P, Ghezzi P, Salio M, Cerami A, Brines
M. Recombinant human erythropoietin protects the myocardium from ischemia-reperfusion
injury and promotes beneficial remodeling. Proclaimed National Acadamie of Sciences, USA
2003;100:4802-4806.
35. Moon C, Krawczky M, Ahn D, Ahmet I, Paik K, Lakatta EG, Talan MI. Erythropoietin
reduces myocardial infarction and left ventricular functional decline after coronary artery
ligation in rats. Proclaimed National Acadamie of Sciences, USA 2003;100:11612-11617.
36. Rui T, Cepinskas G, Feng Q, Kvietys PR. Delayed preconditioning in cardiac myocytes
with respect to development of a proinflammatory phenotype: role of SOD and NOS.
Cardiovascular Research 2003;59:901-911.
37. Tada H, Kagaya Y, Takeda M, Ohta J, Asaumi Y, Satoh K, Ito K, Karibe A, Shirato K,
Minegishi N, Shimokawa H. Endogenous erythropoietin system in non-hematopoietic lineage
cells play a protective role in myocardial ischemia/reperfusion. Cardiovascular Research
2006;71:466-477.
38. Bienvenu AL, Ferrandiz J, Kaiser K, Latour C, Picot S. Artesunateerythropoietin
combination for murine cerebral malaria treatment Acta Trop 2008;106:104-108
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39. Digicaylioglu M, Lipton SA, Erythropoietin-mediated neuroprotection involves cross-talk
between Jak2 and NF-kappaB signalling cascades. Nature 2001;412:601-602.
40. Chen J, Connor KM, Aderman CM, Willett KL, Aspegren OP, Smith LE. Supression of
retinal neovascularization by erythropoietin siRNA in a mouse model of proliferative
retinopathy. Invest Ophthalmol Vis Sci 2009;50:1329- 1335.
41. Morita M, Ohneda O, Yamashita T, Takahashi S, Suzuki N, Nakajima O, Kawauchi S,
Ema M, Shibahara S, Udono T, Tomita K, Tamai M, Sogawa K, Yamamoto M, Fujii-
Kuriyama Y. HLF/HIF-2alpha is a key factor in retinopathy of prematurity in association with
erythropoietin. EMBO Journal
2003;22:1134-1146.
42. Friedman EA, Brown CD, Berman DH. Erythropoietin in diabetic macular edema and
renal insufficiency. American Journal of Kidney Diseases 1995;26:202-208.
43. Lee IG, Chae SL, Kim JC. Involvement of circulating endothelial progenitor cells and
vasculogenic factors in the pathogenesis of diabetic retinopathy. Eye 2006;20:546-552.
44. Tong Z, Yang Z, Patel S, Chen H, Gibbs D, Yang X, Hau VS, Kaminoh Y, Harmon J,
Pearson E, Buehler J, Chen Y, Yu B, Tinkham NH, Zabriskie NA, Zeng J, Luo L, Sun JK,
Prakash M, Hamam RN, Tonna S, Constantine R, Ronquillo CC, Sadda S, Avery RL, Brand
JM, London N, Anduze AL, King GL, Bernstein PS, Watkins S, Genetics of Diabetes and
Diabetic Complication Study Group, Jorde LB, Li DY, Aiello LP, Pollak MR, Zhang K.
Promoter polymorphism of the erythropoietin gene in severe diabetic eye and kidney
complications. Proclaimed National Acadamie of Sciences, USA 2008;105:6998-7003.
45. Manzoni P, Maestri A, Gomirato G, Takagi H, Watanabe D, Matsui S. Erythropoietin as a
retinal angiogenic factor in proliferative diabetic retinopathy. New England Journal of
Medicine 2005;353:782-792
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46. Holstein JH, Menger MD, Scheuer C, Meier C, Culemann U, Wirbel RJ, Garcia P,
Pohlemann T. Erythropoietin (EPO): EPO-receptor signaling improves early endochondral
ossification and mechanical strength in fracture healing. Life Science 2007;80:893-900.
47. Gough NR. Making Sense of EPO Receptors. Science 2008;320:1397 48. Foster DJ, Moe
OW, Hsia CCW Upregulation of erythropoietin receptor during postnatal and
postpneumonectomy lung growth. Am J Physiol 2004;287:L1107L1115.
49. Brines M, Cerami A. Emerging boilogicala roles for erythropoietin in the nervous system.
National Review on Neuroscience 2005;6:484-894.
50. Bod E, Kromminga A, Funk W, Laugsch M, Duske U, Jelkmann W, Paus R. Human hair
follicles are an extrarenal source and a nonhematopoietic target of erythropoietin. FASEB J
2007; 21:3346-54.
51. Paus R, Bod E, Kromminga A, Jelkmann W. Erythropoietin and the skin: a role for
epidermal oxygen sensing? Bioessays 2009; 31:344-8.
52. Ito M, Liu Y, Yang Z, Nguyen J, Liang F, Morris RJ, Cotsarelis G. Stem cells in the hair
follicle bulge contribute to wound repair but not to homeostasis ofthe epidermis. Nature
Medicine 2005;111351-1354.
53. Maxwell PH, Ferguson DJ, Nicholls LG, Johnson MH, Ratcliffe PJ. Theinterstitial
response to renal injury: fibroblast-like cells show phenotypic changes and have reduced
potential for erythropoietin gene expression.
Kidney International 1997;52:715-724.
54. Boutin AT, Weidemann A, Fu Z, Mesropian L, Gradin K, Jamora C, Wiesener M, Eckardt
KU, Koch CJ, Ellies LG, Haddad G, Haase VH, Simon MC, Poellinger L, Powell FL,
Johnson RS. Epidermal sensing of oxygen is essential for systemic hypoxic response. Cell
2008;133:223-34.
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8/7/2019 Rejuvenation Research_Role of Trauma_accepted
24/31
ForPeer
Review
55. Kchling J, Curtin PT, Madan A. Regulation of human erythropoietin gene induction by
upstream flanking sequences in transgenic mice. Br. J. Haematol 1998;103:9608.
56. Higley HR, Ksander GA, Gerhardt CO, Falanga V. Extravasation of macromolecules and
possible trapping of transforming growth factor-beta in venous ulceration. British Journal of
Dermatology 1995;132:79-85.
57. Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes: estimates
for the year 2000 and projections for 2030. Diabetes Care2004; 27:1047-1053,
58. Brem H, Tomic-Canic M. Cellular and molecular basis of wound healing in diabetes. J
Clin Invest. 2007;117:1219-1222.
59. Falanga V. Wound healing and its impairment in the diabetic foot. Lancet 2005;
366:1736-1743.
60 . Langer A, Rogowski W.Systematic review of economic evaluations of human cell-
derived wound care products for the treatment of venous leg and diabetic foot ulcers .BMC
Health Serv Res. 2009 ;9:115.
61. Hanft JR, Surprenant MS. Healing of chronic foot ulcers in diabetic patients treated with a
human fibroblast-derived dermis. J Foot Ankle Surg. 2002 ;41:291-299.
62. A. Hjerppe, M. Hjerppe, V. Autio, R. Raudasoja, A. Vaalasti, . Treatment of Chronic
Leg Ulcers with a Human Fibroblast-Derived Dermal Substitute: A Case Series of 114
Patients. Wounds. 2004;16(3) 2004 Health Management Publications, Inc
63. Brines M, Cerami A.Erythropoietin-mediated tissue protection: reducing collateral
damage from the primary injury response. J Intern Med. 2008;264:405-432.
64. Hamed S, Ullmann Y, Masoud M, Hellou E, Khamaysi Z, Teot L.Topical erythropoietin
promotes wound repair in diabetic rats.J Invest Dermatol. 2010;130287-94.
65. Rappolee DA,Mark D, Banda MJ, Werb Z.Wound macrophages express TGF-alpha and
other growth factors in vivo: analysis by mRNA phenotyping.Science. 1988 ;241:708-712.
ge 23 of 29 Rejuvenation Research
http://www.ncbi.nlm.nih.gov/pubmed/17476353http://www.ncbi.nlm.nih.gov/pubmed?term=%22Langer%20A%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Rogowski%20W%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Hanft%20JR%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Surprenant%20MS%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Brines%20M%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Cerami%20A%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Hamed%20S%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Ullmann%20Y%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Masoud%20M%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Hellou%20E%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Khamaysi%20Z%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Teot%20L%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Rappolee%20DA%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Mark%20D%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Banda%20MJ%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Werb%20Z%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Werb%20Z%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Banda%20MJ%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Mark%20D%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Rappolee%20DA%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Teot%20L%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Khamaysi%20Z%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Hellou%20E%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Masoud%20M%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Ullmann%20Y%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Hamed%20S%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Cerami%20A%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Brines%20M%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Surprenant%20MS%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Hanft%20JR%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Rogowski%20W%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Langer%20A%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/174763538/7/2019 Rejuvenation Research_Role of Trauma_accepted
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66.Mikhal'chik EV, Piterskaya JA,Budkevich LY,Pen'kov LY,Facchiano A,De Luca C,
Ibragimova GA, Korkina LG.Comparative study of cytokine content in the plasma and wound
exudate from children with severe burns. Bull Exp Biol Med. 2009 ;148:771-775.
67. Rezaeian F, Wettstein R, Amon M, Scheuer C, Schramm R, Menger MD, Pittet B, Harder
Y. Erythropoietin protects critically perfused flap tissue. Ann Surg. 2008 ;248:919-929.
68. Galeano M, Altavilla D, Cucinotta D, Russo GT, Cal M, Bitto A, Marini H, Marini R,
Adamo EB, Seminara P, Minutoli L, Torre V, Squadrito F. Recombinant human
erythropoietin stimulates angiogenesis and wound healing in the genetically diabetic mouse.
Diabetes. 2004 ;53:2509-2517.
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Figure 1
Cells were cultured from human biopsies and grown to the 4th passage in vitro. (a) The
mRNA expression profile of the EPO receptor in FmSCs. Sequencing of the PCR product for
the mRNA of the EPO receptor showed 90-98% sequence homology. (b, c) Proliferation of
FmSCs under hypoxic conditions and under the influence of 10 ng/ml trauma cytokine
stimulation. (b) Stimulation with EPO alone in the absence of any additional cytokines under
otherwise identical cell culture conditions showed an inhibitory effect on stem cell growth. (c)
IL-6 stimulation of FmSCs also resulted in decreased growth activityin vitro
. This was
reversed in the presence of EPO. Each point represents the mean SD of nine experiments
and statistically significant difference (P < 0.005, students test).
Figure 2
Phenotype of the FmSCs, determined using flow cytometry. EPO triggering did not change
the phenotype at all; CD31, CD45, CD 73 remained stable with or without the presence of
trauma cytokines. No major population shift.
Figure 3
Phenotype of FmSCs did not change with CD105 and CD166 remained stable with or without
the presence of trauma cytokines. We did observe that cells expressing CD90 partially
switched to a non-CD90-expressing subpopulation in the presence of IL-6. This IL-6 effect
paralleled the growth curves in the presence of IL-6. No major population shift except this
(Control CD 166: EPO CD 166).
Figure 4
(a)Patient A was a 26 year male, who had suffered 25 % body surface flame burn injury,
requiring split skin grafting on day 7 after trauma.A 26-year-old patient with a 0.3-mm split-
thickness skin graft donor site seven days after three treatment sessions without (left) and with
local erythropoietin (right). The polyurethane film dressing was not changed until removal
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seven days following surgery, which allowed a standardized wound management and a
comparability of the healing results. Note the closed, dry and pale-red wound surface of the
EPO treated side indicating a complete reepithelization as well as the secretion and the dark-
red surface of the non-EPO donor site as a sign of incomplete healing.
(b) Patient B was a 64 year lady with a pressure sore (Campbell stage VI including
deperiosted calcanear bone) of her right heel following urosepsis. Sural flap plasty had been
performed already with partial flap loss. The patient had refused further reconstructive
procedures but was focused on preventing amputation by all conservative means. A 64-year-
old diabetic patient with a partial necrotic heel after urosepsis providing poor granulation
tissue following conventional treatment (left). Clinical results after five treatments with local
EPO and subsequent split-thickness skin grafting (right). Note the almost complete wound
closure. The preoperative preparation of the wound allowed salvage of the limb by a minor
surgical procedure.
(c) Patient C was a 69 year male with peripheral arterial occlusive disease, stage IV with a
single arterial supply for the lower leg, non suitable for interventional or surgical
macrovascular reconstruction. The patient had distal leg ulcer and partial necrosis of the
peroneal tendons since more than 6 months. A 69-year-old diabetic patient with a grade III
ulcer at the lateral malleolus based on a peripheral arterial disease grade IV. Exposed tendons
at the bottom of the wound and absence of granulation tissue formation subsequent
revascularization and conservative wound treatment (left). Clinical results after only five local
treatments with EPO optimizing the wound bed for subsequent split-thickness skin grafting
(right). Note the complete wound closure, which has been stable for more than 12 months.
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