Clinical Hemorheology and Microcirculation 61 (2015) 279–290 DOI 10.3233/CH-151982 IOS Press

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Platelet-rich plasma stimulates dermal microvascular endothelial cells and adipose derived stem cells after external radiation F. Haubnera,1,∗ , D. Muschtera,1 , N. Schustera , F. Pohlb , N. Ahrensc , L. Prantld and H.G. Gassnera a

Department of Otorhinolaryngology, Division of Facial Plastic Surgery, University of Regensburg, Germany b Department of Radiotherapy, University of Regensburg, Germany c Department of Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany d Center for Plastic, Aesthetic, Hand & Reconstructive Surgery, University Hospital Regensburg, Regensburg, Germany Abstract. BACKGROUND: Platelet-rich plasma (PRP) products are currently suggested in the treatment of chronic wounds due to possible pro-angiogenic effects. Microvascular compromise represents the major component in radiogenic wound healing complications. The effects of PRP on irradiated cells of the cutaneous wound healing process are still poorly understood. MATERIAL AND METHODS: Human dermal microvascular endothelial cells (HDMEC) and human adipose derived stem cells (hASC) were cultured and irradiated with doses of 2 to 12 Gy. PRP was activated, characterized and added to the incubation media in different concentrations after external radiation. Cell count was determined 48 h after radiation using a semi-automated cell counting system. Levels of interleukin-6 (IL-6), basic fibroblast growth factor (bFGF) and soluble intercellular adhesion molecule-1 (sICAM-1) in the supernatants of HDMEC and hASC co-cultures were determined by enzyme-linked immunosorbent assay (ELISA). Non-irradiated hASC and HDMEC served as controls. RESULTS: The employed PRP preparations were characterized and contained platelet derived growth factor (PDGF-AB), vascular endothelial growth factor (VEGF), bFGF and high levels of sICAM-1. Addition of PRP to irradiated cultures of HDMEC and hASC prevented profound radiation-induced decline in cell numbers. 10% PRP restored cell numbers to levels of untreated, non-irradiated cultures. Basic FGF expression was decreased significantly in hASC monocultures treated with 10% PRP without external radiation and after irradiation with 6 and 12 Gy. These inhibitory effects of PRP were also observed in HDMEC. In contrast, co-cultures of HDMEC-ASC showed a dose-dependent increase in bFGF expression when treated with 5 or 10% PRP. Doses of 6 and 12 Gy increased IL-6 expression in cultures stimulated with 5% PRP. CONCLUSION: Use of PRP in co-cultures of hASC and HDMEC restores proliferative defects caused by external radiation probably by induction of bFGF. Under irradiated conditions, PRP might induce pro-inflammatory stimuli which could be beneficial in treatment of chronic wounds where healing processes are defective. Combined use of hASC and PRP products might be helpful in the treatment of radiogenic wounds. Keywords: Microvascular endothelial cells, human adipose-derived stem cells, endothelial dysfunction, cytokines, adhesion molecules, growth factors, radiation therapy

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Both authors contributed equally to this work. Corresponding author: Frank Haubner, M.D., Department of Otorhinolaryngology, University of Regensburg, Germany, Franz-Josef-Strauß-Allee 11, 93053 Regensburg, Germany. Tel.: +49 941 9449415; Fax: +49 941 9449515; E-mail: [email protected]. ∗

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F. Haubner et al. / Platelet-rich plasma stimulates dermal microvascular endothelial cells

1. Introduction Compromised wound healing after external radiation represents a major clinical challenge [20]. Endothelial dysfunction is an important contributor to compromised wound healing in this context, manifesting in atherosclerosis, fibrosis and vascular occlusion [11]. Early endothelial damage has been identified as a possible target for new therapeutic interventions. One previous study of our group suggested important effects of external radiation on human dermal microvascular endothelial cell (HDMEC) function, showing that proliferation was impaired and levels of different cytokines and adhesion molecules were significantly elevated by external radiation [19]. These effects may be of important clinical relevance in the pathogenesis of radiation-induced wound healing complications. In a follow-up-study, our group examined possible radiation-induced alterations in a co-culture model of human adipose-derived stem cells (hASC) and HDMEC [18]. Here, co-culture of HDMEC with hASC mitigated the increased expression of cytokines and adhesion molecules. There are conflicting reports regarding the treatment of chronic wounds with platelet-rich plasma [8, 10]. Hadad et al. reported improved wound healing in irradiated pig skin with a combination therapy approach, using ASC and platelet-rich plasma (PRP) [15]. The most recent Cochrane review concluded that there is currently not enough evidence to suggest that PRP alone is of value for treating chronic wounds, based on a small number of randomized controlled trials [32]. Concerning the effect of PRP products on irradiated tissues, only a limited number of studies were identified. These publications documented optimized wound healing conditions after the application of PRP products in patients with osteoradionecrosis [1, 38]. Within the phases of wound healing, platelets are activated by contact with collagen or body foreign surfaces, exposed to the bloodstream after endothelial injury [23]. Platelets secrete stored intercellular mediators and cytokines from the cytoplasmic pool and release their ␣-granule content after activation [4]. Currently, seven fundamental protein growth factors have been proven to exist within ␣-granules for initiating wound healing [29]. These growth factors include the isomers of platelet-derived growth factor (PDGF), transforming growth factor-beta (TFG-␤) and the vascular endothelial growth factor (VEGF) [2, 27]. Tissue regeneration may be supported by stimulating effects of PRP concerning cell growth and neoangiogenesis [33]. Based on the results of our previous study on irradiated microvascular endothelial cells and the therapeutic potential of adipose-derived stem cells in cutaneous wound healing, we evaluated the effects of PRP on these cells after external radiation.

2. Material and methods 2.1. Endothelial cell culture Human dermal microvascular endothelial cells (HDMEC, adult donor, PromoCell, Heidelberg, Germany) were maintained in endothelial cell growth medium MV (PromoCell, Heidelberg, Germany), the culture incubator was set at 37◦ C with 5% carbon dioxide. For long-term storage cells were frozen in liquid nitrogen in optimized cryo medium (Cryo-SFM, C-29910, Promocell, Heidelberg, Germany). Cells were used for experiments at passage 3–6.

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2.2. Adipose-derived stem cell culture Human adipose-derived stem cells (hASC, isolated as previously described by Gehmert et al. [12]) were maintained in hASC medium consisting of ␣MEM containing 10% fetal calf serum, 2 mM L-glutamine and 1% penicillin/streptomycin (Sigma, St. Louis, MO, USA) at 5% CO2 and 37◦ C. For long-term storage, cells were frozen in liquid nitrogen in aMEM with 40% fetal calf serum and 10% DMSO. For experiments, cells were used from passage 3 to 7. Briefly, subcutaneous fat tissue from patients undergoing selective body-contouring procedures was washed in phosphate-buffered saline and minced into pieces

Platelet-rich plasma stimulates dermal microvascular endothelial cells and adipose derived stem cells after external radiation.

Platelet-rich plasma (PRP) products are currently suggested in the treatment of chronic wounds due to possible pro-angiogenic effects. Microvascular c...
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