Rejuvenation Research

Rejuvenation Research
Rejuvenation Research: http://mc.manuscriptcentral.com/rejuvenationresearch
Role of trauma cytokines and erythropoietin

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and their therapeutic potential for acute and chronic
wounds
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Journal: Rejuvenation Research

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Manuscript ID: REJ-2010-1050.R1
Manuscript Type: Clinical Articles

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Date Submitted by the

02-Jul-2010
Author:
Complete List of Authors: Bader, Augustinus; Biotechnological Biomedical Center, Department

Re
of Cell Techniques and Stem Cell Biology, University of Leipzig

Lorenz, Katrin; Biotechnological Biomedical Center, Department of
Cell Techniques and Stem Cell Biology, University of Leipzig
Richter, Anja; Biotechnological Biomedical Center, Department of
vi
Scheffler, Katja; Biotechnological Biomedical Center, Department of

Kern, Larissa; Biotechnological Biomedical Center, Department of
ew

Ebert, Sabine; Biotechnological Biomedical Center, Department of
Giri, shibashish; Biotechnological Biomedical Center, Department of
Behrens, Maria; Medical Writing Experts
Dornseifer, Ulf; Klinikum Bogenhausen, Department of Plastic,
Reconstructive, Hand and Burn Surgery
Macchiarini, Paolo; Hospital Clinico de Barcelona, Barcelona,
4Department of General Thoracic Surgery
Machens, Hans-Gunther; Klinikum Rechts der Isar, Technische
Universität München, 5Department of Plastic and Hand Surgery
Regeneration, Skin Aging, Stem Cells, Quality of Life, Growth

Keyword:
Factors
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Page 1 of 29 Rejuvenation Research
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3 The interactive role of trauma cytokines and erythropoietin
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6 and their therapeutic potential for acute and chronic wounds
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Augustinus Bader1, Katrin Lorenz1, Anja Richter1, Katja Scheffler1, Larissa Kern1, Sabine
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13 Ebert1, Shibashish Giri1, Maria Behrens2, Ulf Dornseifer3, Paolo Macchiarini4, Hans-Günther
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15 Machens5
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University of Leipzig, Centre for Biotechnology and Biomedicine, Department of Applied
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20 Stem Cell Biology and Cell Techniques, Germany
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Medical Writing Experts, Langwedel, Germany
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Klinikum Bogenhausen, Zentrum für Schwerbrandverletzte, München
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27 Hospital Clinico de Barcelona, Dept. of General Thoracic Surgery
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Klinik für Plastische Chirurgie, Klinikum Rechts der Isar, Technische Universität München,
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Abstract:
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40 If controllable, stem cell activation following injury has the therapeutic potential for
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supporting regeneration in acute or chronic wounds. Human dermally derived stem cells
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45 (FmSCs) were exposed to the cytokines IL-6, IL-1ß and TNF-α in the presence of
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47 erythropoietin. Cells were cultured under ischemic conditions and phenotypically
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50 characterized using flow cytometry. Topical EPO application was performed in three
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52 independent clinical wound healing attemps. The FmSCs expressed the receptor for
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54 erythropoietin (EPO). EPO had a strong inhibitory effect on FmSC growth in the absence of
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57 IL-6 and TNF-α. With IL-6, the EPO effects were reversed to that of growth stimulation.
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59 TNF-α had the strongest stimulatory effect. In contrast, IL-1ß had an inhibitory effect.
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Topically applied EPO considerably enhanced wound healing and improved wound
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3 conditions of acute and chronic wounds. Site specificity of stem cell activation is mediated by
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6 IL-6 and TNF-α. In trauma, EPO ceases its inhibitory role and reverts to a clinically relevant
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8 boosting function. EPO may be an important therapeutic tool for the topical treatment of acute
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and chronic wounds.
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15 INTRODUCTION
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The human body has command of a tool box that allows it to appropriately react to injury with
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20 an amazing site specificity to achieve a regenerative response. It has been unclear how local
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22 stem cells are “awakened” in such instances of need. If this mechanism was better understood,
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a fundamental therapeutic strategy that would allow site-specific stem cell activation in any
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27 area of the human body could be developed. Site specificity of tissue regeneration has been
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taken for granted as a “normal” process, but their underlying mechanisms, relevance for stem
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32 cell-based responses and therapeutic potential have not yet been understood. Frequently, this
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34 innate capacity is not sufficient to lead to full restoration, resulting in so-called “nonhealing”
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wounds.
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39 It is well-known that local trauma leads to the release of inflammatory cytokines, including
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41 IL-6, IL-1β and TNF-α.1 Mesenchymal stem cells (MSCs), isolated from many human tissues,
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such as bone marrow, adipose tissue, the adult liver, peripheral blood, amniotic fluid, the
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46 bronchial lung, the articular synovium and other fetal tissues, are a cell population that
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48 possesses a fibroblastic-like morphology, limited but long-term viability, self-renewal
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51 capacity and multilineage potential.2,3 They are characterized by similar surface antigen
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53 expression patterns for CD14(-), CD31(-), CD34(-), CD45(-), CD71(+), CD73/SH3-SH4(+),
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55 CD90/Thy-1(+), CD105/SH2(+), CD133(-) and CD166/ALCAM(+)4,5. MSCs can
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58 differentiate into adipogenic, osteogenic, myogenic, chondrogenic and neurogenic cell types.
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3 In an effort to identify new sources of mesenchymal stem cells, dermal rodent fibroblast cell
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6 lines were examined for their mesenchymal potential.9-11 Publications by Toma et al. 9
and
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8 Crigler et al. suggest that the adult mammalian dermis contains tissue-derived stem cells
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and that these fibroblastic mesenchymal stem cells are more plastic than previously
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13 appreciated. Dermis-derived fibroblastic mesenchymal stem cells have been used for
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15 therapeutic applications such as transplantation to support bone formation.12-14 Toma et al.9
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demonstrated the mesenchymal plasticity of primary human dermal fibroblasts in vitro with
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20 different approaches regarding characterization and applications.15-18 Zuk et al. analyzed the
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22 phenotypic characteristics of these fibroblasts and noted that their phenotype seems to be
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similar to that of adult-derived stem cells (ADSCs).5,16,18
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To continue these studies, we examined whether unselected human dermis fibroblastic

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32 mesenchymal cells possess stem cell-like characteristics and are phenotypically similar to
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34 ADSCs. Bone marrow-derived MSCs (bmMSCs) have been shown to support the wound
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healing of chronic skin wounds. Badiavas and Falanga (2003) demonstrated that chronic skin
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39 ulcers of patients with arterial and venous insufficiency were healed with complete wound
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41 closure and less scar formation when treated with bmMSC-seeded grafts.21
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46 To characterize FmSCs relative to ADSCs, we analyzed the cytoskeletons and compositions
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48 of the extracellular matrix as well as the mesenchymal phenotypes and differentiation
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51 properties of both. The obtained data show for the first time that primary human dermal
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53 fibroblasts in vitro share common characteristics with ADSCs, such as phenotype and
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55 differentiation potential. Our results demonstrated that FmSCs fulfill the three main
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58 characteristics of MSCs: they express all MSC-related surface antigens homogenously; their
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60 cytoskeleton and matrix compositions are quite similar to that of MSCs; and they differentiate
along the adipogenic and osteogenic cell lineages.20
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6 Both conditions, however, do not sufficiently explain the mechanism of endogenous stem cell
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8 activation in the case of injury alone.
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We therefore developed an in vitro model of trauma conditions by investigating the role of IL-
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13 6, IL-1β and TNF-α on human skin stem cells, identified a receptor for Erythropoietin (EPO)
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15 in these cells and investigated the role of EPO with and without the presence of the trauma
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cytokines. The knowledge obtained from these studies was then transferred to three
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20 independent and specific clinical cases: EPO was topically applied to a split-thickness skin
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22 graft donor site, a pressure and a vascular ulcer.
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27 EPO has been used in clinical practice for a wide range of diseases22, obtaining systemic
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application via subcutaneous, intramuscular or intravenous route.23-28 A topical administration

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32 to stem cells at the site of the wound injury would allow a more direct stimulatory response
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34 and would work only if an appropriate interference with site-specific mechanistic responses
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occurred. The elucidation of such mechanisms and the development of a therapeutic potential
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39 has been the scope and success of this study.
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44 METHODS
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48 Cell isolation
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51 Human juvenile foreskin samples were obtained from four-year-old patients undergoing
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53 circumcision after written consent was obtained. This study was approved by the ethics
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55 commission of Leipzig University and was conducted in accordance with the Declaration of
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58 Helsinki protocols.
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60 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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3 tissue was cut into small pieces and washed three times with sterile phosphate-buffered saline
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6 (PBS) at room temperature. The pieces were then incubated with 0.075% collagenase type A
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8 (Roche Diagnostics, Mannheim, Germany) for 12 h at 37°C with gentle agitation.
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For FmSC suspensions, the enzymatic reaction was inactivated with DMEM/10% FBS
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13 (Gibco/Invitrogen, Karlsruhe, Germany) and filtered through a 70-µm mesh. This cell
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15 suspension was centrifuged at 600 x g for 5 min. The cell pellet was then gently resuspended
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in DMEM/10% FBS, filtered through a 70-µm mesh and plated in conventional T75 tissue
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20 culture flasks (BD Falcon, Heidelberg, Germany). Cells were cultured in DMEM
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22 supplemented with 10% FBS, GlutaMAX-I, 4.5 g/L glucose and pyruvate (Gibco/Invitrogen).
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27 Cell proliferation
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FmSCs were seeded in six-well plates at passage 4. After one day of cultivation in
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32 DMEM/10% FBS, the cells attached and adapted to start proliferation. On the following day,
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34 the cells were cultured with the same medium but without FBS to minimize serum-induced
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effects. The next day, cytokine stimulation of the cells was initiated with cultivation in
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39 DMEM/10% FBS supplemented with or without 10 ng/mL EPO in combination with 10
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ng/mL IL-6, 10 ng/mL IL-1β or 10 ng/mL TNF-α. Controls were also performed with each
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cytokine alone. At days 3, 5, 7 and 11, cells were trypsinized and viable cell numbers were
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46 counted in a hemocytometer by trypan blue staining. All experiments were done in triplicate,
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48 with three independent sets of patient materials.
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53 Cell immunophenotyping
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55 Immunophenotyping of FmSCs was performed as described previously by Lorenz et al19. The
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58 following labeling reagents were used: fluorescein isothiocyanate (FITC)- or phycoerythrin
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60 (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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3 human CD105 (BD Biosciences, Heidelberg, Germany) and anti-human CD166 (Acris
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6 Antibodies, Hiddenhausen, Germany). The monoclonal antibody mouse anti-human CD73
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8 (BD Biosience) was unlabeled and combined with a secondary PE-labeled goat anti-mouse
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antibody (Sigma-Aldrich). Incubation and flow cytometry analyses were performed according
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13 to conventional techniques29. Isotype controls were equally concentrated, labeled or
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15 unlabeled. The stained cells were analyzed on a FACS Calibur (BD Bioscience) using
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CellQuest Pro (BD Bioscience). Fluorescence intensities were determined by flow cytometry
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20 in a minimum of 1x104 cells.
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Growth curves
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27 To record a growth curve, three individual tests were performed, each in triplicate. Cell
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counting was performed at day 0, 1, 3, 5 and 7 by trypsinization and trypan blue staining
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32 using a Neubauer’s chamber. The cells were seeded at passage 6 with a density of 20,000
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34 cells/well (9.6 cm2) in a six-well plate (Falcon) two days before stimulation (day 0). The
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exchange of media one day before stimulation from 10% FBS-containing DMEM to serum-
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39 free DMEM ensured the attachment of the cells during the last 24 hours and a minimal protein
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background from the FBS. The stimulation was made with or without the presence of IL-6,
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IL-1β and TNF-α by adding EPO (NeoRecormon, Roche) and/or the cytokines (all
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46 RELIAtech) to the media and performing a full media exchange. At days 3 and 5, half the
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48 volume of the media was changed with media containing the initial concentrations of EPO
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51 and/or cytokine. The cell scores of each sample and the average of all nine samples were
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53 calculated, including the standard deviation. To compare the different growth curves, a
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55 Student’s t-test was used to test the significance of the differences between the breakpoints.
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58 For stimulation with cytokines, a concentration-dependent pretest was performed to identify
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60 the minimum concentration needed for successful stimulation; this was determined to be 10
ng/mL.
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6 Clinical cases
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8 In three highly different clinical situations, topical EPO application was used to support
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wound healing. All patients provided informed consent based on the guidelines of the local
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13 ethical committee and the national legal requirements in Germany. Topical treatment was
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15 performed using a mixture of 3,000 IE erythropoietin-ß (NeoRecormon®, F. Hoffmann-La
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Roche AG, Basel, Schweiz) and 20 g hydrogel (Varihesive®, ConvaTec, NJ, USA). In patient
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20 A, the mixture was topically applied to a 0.3-mm deep split-thickness skin graft donor sites
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22 measuring 8 x 24 cm directly after skin harvesting. The donor site at the thigh was
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27 After three and six days, the mixture was again applied by puncturing the polyurethane film,
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which remained on the wound. At day 7, the film dressing was removed to evaluate the
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32 reepithelialization. Another donor site of equal depth and dimension at the contralateral leg of
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34 patient A was treated similar but the mixture was replaced by hydrogel alone - without EPO.
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37 Again the dressing rest in place for seven days followed by dressing removal and wound
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39 assessment. The standardized wound management provided an ideal comparability of both
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similar wounds.
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44 Patient B had a non-healing pressure sore at the heel following urosepsis and Patient C had a
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46 non-healing vascular ulcer. In both patients the wounds were surgically debrided and treated
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in the same manner, using the same mixture of EPO and hydrogel. Moist wound management
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51 was provided by covering both wounds with Varihesive® (Convatec, Skillman, NJ, USA). A
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53 total of five dressing changes with new EPO applications were performed in both patients to
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prepare the wound for skin grafting. The poor general condition of patient B and C did not
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58 allow extensive reconstructional procedures. Therefore, the aim of local EPO application was
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60 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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3 RESULTS
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6 Sequencing the EPO receptor in human FmSCs
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8 Cells were characterized for their expression of CD31, CD45, CD73, CD90, CD105 and
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CD166. The expression of CD34, CD71 and CD133 was also examined. Fig. 1a shows the
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13 mRNA expression profile of the EPO receptor in FmSCs. Sequencing of the PCR product for
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15 the mRNA of the EPO receptor showed 90-98% sequence homology.
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Figure 1
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20 Figure 2
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22 Figure 3
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32 Switch from inhibitory to stimulatory effects of EPO on stem cell proliferation
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34 Cells were cultured from human biopsies and grown to the 4th passage in vitro. Stimulation of
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EPO alone in the absence of any cytokines showed an inhibitory effect on stem cell growth
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39 (Fig. 1b). Cells under control conditions grew up to 1.545 million cells. In contrast, we
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observed a dramatic decrease in cell proliferation when the FmSCs were stimulated with
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EPO. Cell proliferation was minimized by 32%, to a total cell number of 1.053 million cells.
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46 IL-6 stimulation of FmSCs also resulted in decreased growth activity relative to the control
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48 cells, but this was reversed in the presence of EPO (Fig. 1c). This indicates both an inhibitory
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51 role of EPO (Fig. 1b) in the absence of cytokines or in the late phase of trauma and a
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53 supportive, boosting activity of EPO in the presence of IL-6 (Fig. 1c). TNF-α was a strong
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55 stimulator of FmSC proliferation, and the presence of EPO did not influence this. Cell
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58 proliferation was elevated most with TNF-α. IL-1β had an inhibitory effect on the
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60 proliferation of the stem cells compared to controls cells, both with and without EPO.
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3 Effect of stimulation on the expression of stem cell markers
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6 To characterize the FmSCs, surface antigens were analyzed by flow cytometry. FmSCs
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8 homogenously expressed CD90, CD73, CD105 and CD166. (Fig. 2, Fig. 3) In contrast,
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expression of endothelial cell surface markers such as CD31 was not detected. In addition,
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13 hematopoietic cell subpopulations positive for surface antigens such as CD45, CD14 and
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15 CD133 were not observed.
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During cultivation and stimulation of FmSCs with inflammatory cytokines in combination
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20 with EPO, we did not detect any changes in the surface antigen expression of MSC markers.
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22 Except when cultivating FmSCs with IL-6 in combination with EPO, we found changes in the
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expression of CD90. This suggests that the unselected stem cell population was stimulated
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27 differently, and thus two different CD90-expressing cell populations were detected. (Fig. 3)
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32 In vivo experiments
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34 The more complex in vivo situation is characterized by an intricate interplay of cytokine
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profiles, consisting of mixtures and time-sequence variations with respect to availability.
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In Patient A, complete and stable reepithelialization of the split-thickness skin graft donor
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site, topically treated with EPO, was achieved seven days after the operation. The wound
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46 surface was closed, dry and clearly looked pale. (Fig.4a) In contrast, the donor site at the
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48 contralateral thigh that was treated without EPO showed incomplete reepithelialization at this
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51 time point, indicated by secretion and a dark-red wound surface. (Fig.4a)
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55 In Patient B, sufficient granulation tissue formation was obtained after five local treatment
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58 sessions with EPO, which provided an highly vascularized wound bed for sucessful split-
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60 thickness skin grafting. The ideal prepared wound bed enabled salvage of the limb without
extensive reconstructive procedures (Fig. 4b).
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6 In Patient C, improved healing and sufficient granulation tissue formation was achieved
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8 following five local applications of EPO. (Fig.4c) After subsequent skin grafting at days 21
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and 56, the wound healed well and has remained stable for more than 12 months (Fig. 4c).
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15 DISCUSSION
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20 Erythropoietin is a type I cytokine that was approved by the US Food and Drug
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22 Administration (FDA) in 1989 for the treatment of the anemia of end-stage renal disease.
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Thereafter, erythropoietin has been used for the treatment of a diverse range of diseases,
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27 including cancer28,31,32, heart care erythropoiesis33-37, malaria38, ischemic and degenerative
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damage of neurons40, retinopathy40,41 and diabetic retinopathy42-45. EPO plays a crucial role in
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32 the process of endochondral ossification in bone repair in mice via EPO-receptor expression46.
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34 Gough (2008) 47 supported the concept that understanding the EPO receptors by which EPO
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signaling contributes to organ development provides information on the differentiation of
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39 erythrocytes. Interestingly, Foster et al. (2004) found increased EPO-R protein levels in
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dynamically growing canine lungs after pneumonectomy, suggesting a paracrine role for EPO
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signaling in lung growth and remodeling. This hypothesis may be applicable to other types of
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46 organ repair since EPO and EPO-R are expressed in several organs (e.g., kidney, brain, heart,
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48 muscle and endothelial cells) 49. However, it is now known that EPO and EPO-R are local
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51 products in a wide range of cells that specifically protect other cells from potentially cytotoxic
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53 events and metabolic stress.
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57 50
58 Adding to the evidence of Bodo et al. (2007) that normal human skin expresses EPO and
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60 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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3 action in the case of traumatic skin injuries. In the case of injury and ischemic trauma,
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6 cytokines IL-6, IL-1β and TNF-α are alerted. We demonstrated that the trauma cytokine IL-6
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8 and EPO synergistically up-regulated stem cell growth in the case of hypoxic skin conditions.
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In contrast, without trauma, EPO exerted an inhibitory effect on in vitro skin stem cells. This
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13 effect reverted to stimulation in the presence of IL-6 and TNF- α. Only IL-1β maintained its
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15 inhibitory function with or without EPO. Neither the trauma cytokines nor EPO grossly
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changed the phenotype of the fibroblast precursor cells.
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20 Our findings agree with the observations of Paus et al. (2009)51 that the oxygen sensing skin
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22 response is mediated by skin EPO. In situations of low oxygen, skin EPO and IL-6 are
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alarmed to react to the site-specific injury. This finding compliments the relevance of our data
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27 as a physiological and potentially highly relevant therapeutic strategy for endogenous stem
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cell activation in the case of trauma.

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34 In the human scalp, it was shown that hair follicles expressed EPO at the mRNA and protein
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levels, up-regulated EPO transcription under hypoxic conditions and expressed transcripts of
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39 EPO-R and the EPO stimulatory transcriptional cofactor hypoxia-inducible factor-1α. These
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findings are in line with recent research results that showed that hair follicle-derived
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keratinocytes were a major cell source for reepithelialization during wound healing52 and that
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46 the hair follicle connective tissue sheath was a source of granulation tissue formation53.
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48 Boutin et al. (2008)54 revealed the EPO-connected oxygen sensing functions of the skin and
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51 elucidated how mammalian cells adapt to low oxygen levels by recruiting the skin as a central
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53 coordinator of the systemic response to hypoxia. Hair follicles are able to detect insufficient
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55 oxygen levels, a crucial mechanism of the extremely fast renewing and proliferating cell
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58 population, to regulate its metabolic balance. Using transgenic mice studies, Kochling (1998)
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60 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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3 has been shown that the circulating levels of EPO may increase up to 1,000-fold in response
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6 to hypoxia in the kidney53.
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In the absence of trauma cytokines, EPO down-regulates the proliferation of skin stem cells in
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13 vitro. In the case of traumatic skin hypoxia, IL-6 and TNF-α activate stem cells. Specifically,
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15 in the presence of IL-6, the inhibitory role of EPO is reversed to increase stem cell
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proliferation. This represents an adequate response to a pathophysiological need. The
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20 stimulatory effects of TNF-α are not diminished by the previously inhibitory role of EPO.
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22 Among the trauma cytokines studied here, only IL-1β also exhibited an inhibitory function
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that did not interfere with the original inhibitory role of EPO. In vivo, we observed the net
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27 effect of cytokine and EPO stimulation in acute and chronic wound types. In both cases, the
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regenerative response was boosted qualitatively and quantitatively. By these mechanisms, the
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32 skin trauma EPO system switches from its inhibitory function to a supportive role for
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34 boosting skin regeneration. The inflammatory activity of the wound itself represents a
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permissive situation for the boosting activity of EPO, which seems to reduce the healing time
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39 at split-skin graft donor sites from 10 to 7 days. In the previously non-healing wounds, EPO
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assumes an enabling role that shifts the balance from non-healing to healing and triggers the
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formation of granulation tissue. The expression of EPO-R on the stem cells suggests that this
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46 could be a normal role of EPO that permits the human body to recover from site-specific
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48 tissue damage or injury in any area of the human body. This mode of action has been
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51 demonstrated clinically by the clearly accelerated reepithelialization of the EPO treated donor
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53 site in direct comparison to the non-EPO treatet donor site at the same patient.
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58 Non-healing chronic wounds are at the opposite end of the spectrum of acute wound-healing
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60 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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3 patients suffer from chronic wounds57,58, with many of them suffering from non-healing
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6 chronic wounds59. There are approximately 5.2 million pressure ulcers and 7.6 million venous
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8 ulcers in the world that require treatment every year.
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13 Fibroblasts form granulation tissue via hyper-proliferation. This leads to a normal process of
14
15 not only cellular rebuilding of lost dermal tissue but also reconstitution of the physical barrier
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18
of the basal lamina and the scaffold for revascularization. Large-area wounds do not heal
19
20 within a short time, so the risk of infection and dehydration rises dramatically. Our results
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22 suggest that we can completely alter the wound-healing landscape and have a major impact on
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the care of both acute and chronic wounds. This study provides mechanistic evidence to
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27 support the hypothesis that this novel treatment modality physically modifies the wound
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microenvironment and thereby promotes wound healing in clinical relevant manner.

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32
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34 In a few clinical applications, fibroblasts were used to treat diabetic or venous ulcers 60-62, but
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this methodology remains controversial. We report a mechanism explaining how endogenous
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39 stem cells can be activated locally at the site of a severe wound without necessitating the
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transplantation of exogenous cells. All clinical cases, although diverse in their

42
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44
pathophysiology, were dramatic successes with respect to their respective healing responses.
45
46
47
48 Brines & Cerami described63 that EPO is locally produced in the immediate surrounding area
49
50
51 of a tissue injury to counteract the destructive effects of cytokines such as TNF-α by
52
53 preventing cell apoptosis, thus the development of secondary, proinflammatory cytokine-
54
55 induced injury can be reduced. However, a delicate balance in tissue injury exists between
56
57
58 EPO and proinflammatory cytokines such as TNF-α. Therefore, compensatory EPO
59
60 production by nearby tissue balances the effects of inflammatory mediators and prevents the
further spread of damage.63 Hamed et al. reported that treatment with topical EPO improves
1
2
3 the defect repair of excised wounds in diabetic rats.64 They suggested that vascular VEGF-
4
5
6 induced angiogenesis, enhanced collagen deposition and reduced apoptosis in the diabetic
7
8 wound bed are among the mechanisms that underlie the effects of topical EPO. This work by
9
10
Hamed et al. 64 was the first to investigate the use of topical EPO treatment for wound healing.
11
12
13 However we are the first of topical EPO treatment for acute and chronic wounds patients.
14
15 Although other cytokines such as tumor growth factor-β, monocyte chemoattractant protein-1
16
17
18
and colony-stimulating factor-1 are released from the invading inflammatory cells to the
19
20 wound bed upon skin injury and in chronic wounds65, we selected these cytokines (IL-6 ,
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22 TNF- α and IL-1ß ) because these are leading cytokines which is associated with organ
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24
trauma injury including chronic wound.66
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Despite the beneficial effect on wound repair, one has to assume that doses of EPO are rather
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31 67
32 high within the defect wound and low systemically. Rezaeian et al demonstrated that a
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34 triplicate intraperitoneal dose of 500IU EPO/kg bw over 48 hours did not influence RBC
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36 68
count and Hematocrit, whereas Galeano et al observed a significant increase in RBC count
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39 and hemoglobin after 12 days of daily subcutaneous administration of 400IU EPO / kg bw. In
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compared to this, the EPO concentration of our present clinical study is 50 U (one time) by
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44
topical application of the hydrogel containing EPO in the patients. This concentration (50U) is
45
46 75 times less than other existing dose of various other experimental or clinical models. Using
47
48 this concentration, we are conducting multicenter clinical trials for actute and choric wound
49
50
51 pateints. Everytime fresh hydrogel is prepared and half life of EPO is 48 hours and stable in
52
53 gel up to 12 weeks. There is no systemic effect of treated patients which is main advantages
54
55 of this topical application. We measured red blood cell (RBC) count and haemoglobin,
56
57
58 leukocyte and platelet count of the patients before and after EPO treatment but there were no
59
60 any difference.
1
2
3 Our present investigation may provide a standard supplemental therapy for reducing the
4
5
6 mortality and morbidity associated with chronic wounds, especially in the elderly, the
7
8 disabled and those with diabetes. Especially large-area burn injuries, where the wound closure
9
10
is a race against time, may benefit from the healing accelerating characteristics of EPO. Not
11
12
13 only the burned, debrided and grafted areas but also the skin graft donor sites have to heal in a
14
15 limited time frame. Frequently, donor sites do not rejuvenate for reharvesting as fast as
16
17
18
needed, resulting in graft deficiencies that may lead to further extensive complications with
19
20 fatal outcomes. The targeted clinical areas will improve in the assistance toward accelerating
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22 regeneration of acute and chronic wounds, and endogenous stem cell activation may reduce
23
24
the need for skin grafting of burns.
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References
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31
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Adamo EB, Seminara P, Minutoli L, Torre V, Squadrito F. Recombinant human
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6 Figure 1
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Cells were cultured from human biopsies and grown to the 4th passage in vitro. (a) The
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10 mRNA expression profile of the EPO receptor in FmSCs. Sequencing of the PCR product for
11
12 the mRNA of the EPO receptor showed 90-98% sequence homology. (b, c) Proliferation of
13
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15 FmSCs under hypoxic conditions and under the influence of 10 ng/ml trauma cytokine
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17 stimulation. (b) Stimulation with EPO alone in the absence of any additional cytokines under
18
19 otherwise identical cell culture conditions showed an inhibitory effect on stem cell growth. (c)
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22 IL-6 stimulation of FmSCs also resulted in decreased growth activity in vitro. This was
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24 reversed in the presence of EPO. Each point represents the mean ± SD of nine experiments
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26 and statistically significant difference (P < 0.005, student’s test).
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31 Figure 2
32 Phenotype of the FmSCs, determined using flow cytometry. EPO triggering did not change
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34 the phenotype at all; CD31, CD45, CD 73 remained stable with or without the presence of
35
36 trauma cytokines. No major population shift.
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Figure 3
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Phenotype of FmSCs did not change with CD105 and CD166 remained stable with or without
42
43 the presence of trauma cytokines. We did observe that cells expressing CD90 partially
44
45 switched to a non-CD90-expressing subpopulation in the presence of IL-6. This “IL-6 effect”
46
47
paralleled the growth curves in the presence of IL-6. No major population shift except this
48 (Control CD 166: EPO CD 166).
49
50
51
52
Figure 4
53
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55 (a) Patient A was a 26 year male, who had suffered 25 % body surface flame burn injury,
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57 requiring split skin grafting on day 7 after trauma.A 26-year-old patient with a 0.3-mm split-
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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
1
2
3 seven days following surgery, which allowed a standardized wound management and a
4
5
6 comparability of the healing results. Note the closed, dry and pale-red wound surface of the
7
8 EPO treated side indicating a complete reepithelization as well as the secretion and the dark-
9
10
red surface of the non-EPO donor site as a sign of incomplete healing.
11
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13
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15 (b) Patient B was a 64 year lady with a pressure sore (Campbell stage VI including
16
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deperiosted calcanear bone) of her right heel following urosepsis. Sural flap plasty had been
19
20 performed already with partial flap loss. The patient had refused further reconstructive
Fo
21
22 procedures but was focused on preventing amputation by all conservative means. A 64-year-
23
24
old diabetic patient with a partial necrotic heel after urosepsis providing poor granulation
rP
25
26
27 tissue following conventional treatment (left). Clinical results after five treatments with local
28
29
ee
EPO and subsequent split-thickness skin grafting (right). Note the almost complete wound
30
31
32 closure. The preoperative preparation of the wound allowed salvage of the limb by a minor
rR
33
34 surgical procedure.
35
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37
ev
38
39 (c) Patient C was a 69 year male with peripheral arterial occlusive disease, stage IV with a
40
41
iew
single arterial supply for the lower leg, non suitable for interventional or surgical
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44
macrovascular reconstruction. The patient had distal leg ulcer and partial necrosis of the
45
46 peroneal tendons since more than 6 months. A 69-year-old diabetic patient with a grade III
47
48 ulcer at the lateral malleolus based on a peripheral arterial disease grade IV. Exposed tendons
49
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51 at the bottom of the wound and absence of granulation tissue formation subsequent
52
53 revascularization and conservative wound treatment (left). Clinical results after only five local
54
55 treatments with EPO optimizing the wound bed for subsequent split-thickness skin grafting
56
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58 (right). Note the complete wound closure, which has been stable for more than 12 months.
59
60
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Fo
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r
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Pe
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er
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Re
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vi
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ew
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209x297mm (150 x 150 DPI)
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Fo
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r
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Pe
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er
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Re
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vi
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ew
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49 209x297mm (150 x 150 DPI)
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Fo
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r
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Pe
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er
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Re
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vi
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ew
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49 209x297mm (150 x 150 DPI)
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Fo
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r
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Pe
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er
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Re
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vi
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ew
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47 146x202mm (72 x 72 DPI)
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Rejuvenation Research - Role of Trauma - Accepted

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Rejuvenation Research: http://mc.manuscriptcentral.com/rejuvenationresearch

Role of trauma cytokines and erythropoietin

Journal: Rejuvenation Research

Manuscript ID: REJ-2010-1050.R1

Manuscript Type: Clinical Articles

Date Submitted by the

Complete List of Authors: Bader, Augustinus; Biotechnological Biomedical Center, Department

of Cell Techniques and Stem Cell Biology, University of Leipzig

Scheffler, Katja; Biotechnological Biomedical Center, Department of

Cell Techniques and Stem Cell Biology, University of Leipzig

Regeneration, Skin Aging, Stem Cells, Quality of Life, Growth

To continue these studies, we examined whether unselected human dermis fibroblastic

along the adipogenic and osteogenic cell lineages.20

application via subcutaneous, intramuscular or intravenous route.23-28 A topical administration

25 subsequently closed with a polyurethane dressing (OpSite, Smith&Nephew, London, UK).

avoid lower leg amputation by a minor surgical procedure.

extensive reconstructive procedures (Fig. 4b).

cell activation in the case of trauma.

microenvironment and thereby promotes wound healing in clinical relevant manner.

transplantation of exogenous cells. All clinical cases, although diverse in their

pharmacokinetics and efficacy in patients with Parkinson's disease. J Neural Transm

Biol Cell 2002;13:4279-4295.

27. Vaccaro M, Magaudda L, Cutroneo G, Trimarchi F, Barbuzza O, Guarneri B. Changes in

combination for murine cerebral malaria treatment Acta Trop 2008;106:104-108.

renal insufficiency. American Journal of Kidney Diseases 1995;26:202-208.

60 . Langer A, Rogowski W.Systematic review of economic evaluations of human cell-

62. A. Hjerppe, M. Hjerppe, V. Autio, R. Raudasoja, A. Vaalasti, . Treatment of Chronic

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