rejuvenation research_role of trauma_accepted

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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

Rejuvenation Research


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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

Page 2Rejuvenation Research


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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.

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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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