49 www.ecmjournal.org F Duttenhoefer et al. EPC and MSC co-culture promotes neovascularisation European Cells and Materials Vol. 26 2013 (pages 49-65) ISSN 1473-2262 Abstract Blood supply is a critical issue in most tissue engineering approaches for large defect healing. As vessel ingrowth from surrounding tissues is proven to be insuficient, current strategies are focusing on the neo-vascularisation process. In the present study, we developed an in vitro pre- vascularised construct using 3D polyurethane (PU) scaffolds, based on the association of human Endothelial Progenitor Cells (EPC, CD34+ and CD133+) with human Mesenchymal Stem Cells (MSC). We showed the formation of luminal tubular structures in the co-seeded scaffolds as early as day 7 in culture. These tubular structures were proven positive for endothelial markers von Willebrand Factor and PECAM-1. Of special significance in our constructs is the presence of CD146-positive cells, as a part of the neovasculature scaffolding. These cells, coming from the mesenchymal stem cells population (MSC or EPC-depleted MSC), also expressed other markers of pericyte cells (NG2 and αSMA) that are known to play a pivotal function in the stabilisation of newly formed pre- vascular networks. In parallel, in co-cultures, osteogenic differentiation of MSCs occurred earlier when compared to MSCs monocultures, suggesting the close cooperation between the two cell populations. The presence of angiogenic factors (from autologous platelet lysates) in association with osteogenic factors seems to be crucial for both cell populations’ cooperation. These results are promising for future clinical applications, as all components (cells, growth factors) can be prepared in an autologous way. Keywords: Mesenchymal stem cells; endothelial progenitor cells; bone; neovascularisation; 3D; co-cultures; autologous; platelet lysates. *Address for correspondence: Sophie Verrier AO Research Institute Clavadelerstrasse 8 CH-7270 Davos, Switzerland Telephone Number: +41 81 414 2448 FAX Number: +41 81 414 2288 Email: sophie.verrier@aofoundation.org Introduction Bone is a highly vascularised tissue that generally leads to spontaneous regeneration of shape and function without scar formation (Salgado et al ., 2004). During bone remodelling and repair, new capillaries derived from pre-existing neighbour vessels invade the site through angiogenesis (Carano and Filvaroff, 2003). Thus, the best healing rates of injuries occur mainly in areas with the highest level of vascularisation (Deleu and Trueta, 1965; Novosel et al., 2011). In cases of large bone defects, not only is the bone tissue damaged, but the surrounding vasculature is often notably debilitated, impairing adequate levels of oxygen and nutrient supply at the injury site and ultimately proper healing (Rhinelander, 1965; Johnson et al., 2011). With a growth rate of around 5 µm/h, sprouting-out from existing vessels is limited in space, and a time-consuming process that requires the presence of blood vessels in the immediate surroundings (Laschke and Menger, 2012). Bone repair highly depends on the presence of osteogenic cells at the healing site (Mandracchia et al., 2001; Carano and Filvaroff, 2003), and bone marrow is a natural reservoir of skeletal stem cells. Recruitment, proliferation and differentiation of MSCs into mature osteoblasts are regulated by many factors including cytokines, growth factors and systemic hormones. These factors are released not only by osteoblastic cells themselves (Lian and Stein, 1995), but also by cells that are part of the tightly connected vascular system, such as endothelial cells (Wang et al., 1997; Street et al., 2002; Villars et al., 2002; Fuchs et al., 2007) and pericytes (Jones et al., 1995). Many studies underline the close relationship between angiogenesis and ontogenesis. Cross-talk between endothelial cells and osteoblasts has been reported, through secreted factors (e.g., VEGF) and through direct cell-cell interactions (e.g., cell surface proteins, gap junctions) (Villars et al., 2000; Meury et al., 2006; Grellier et al., 2009; Santos et al., 2009). Based on this knowledge, contemporary advances focus on neovascularisation of tissue-engineered bone implants (Raii and Lyden, 2003; Unger et al., 2007; Hofmann et al., 2008; Laschke et al., 2008; Fuchs et al., 2009; Capobianco et al., 2010). Postnatal neovascularisation is thought to exclusively result from proliferation, migration, and remodelling of fully differentiated endothelial cells (EC) sprouting from 3D SCAFFOLDS CO-SEEDED WITH HUMAN ENDOTHELIAL PROGENITOR AND MESENCHYMAL STEM CELLS: EVIDENCE OF PREVASCULARISATION WITHIN 7 DAYS F. Duttenhoefer 1,2 , R. Lara de Freitas 1,3 , T. Meury 1,4 , M. Loibl 1,5 , L.M. Benneker 6 , R.G. Richards 1 , M. Alini 1 and S. Verrier 1, * 1 AO Research Institute Davos, Davos, Switzerland 2 Department of Oral and Maxillofacial Surgery, Albert-Ludwigs-University, Freiburg, Germany 3 Medical School of Ribeirão Preto, University of São Paulo, São Paulo, Brazil 4 Roche, Basel, Switzerland 5 Department of Trauma Surgery, Regensburg University Medical Centre, Regensburg, Germany 6 Department of Orthopaedic Surgery, Inselspital, University of Bern, Switzerland