The objective of this work

The objective of this work was to investigate if a synergistic coculture system of MSCs with endothelial Tubastatin A Supplier could affect the number and state of differentiation of harvested MSCs. In contrast to other papers, in this cell-cell communication topic, the investigation was carried out for a longer time, 3weeks instead of 1 to 6days and different ratios of both cell types were considered (Guillotin et al., 2004; Villars et al., 2002; Grellier et al., 2009a; Guillotin et al., 2008; Finkenzeller et al., 2006; Stahl et al., 2004; Wenger et al., 2004; Villars et al., 2000; Xue et al., 2009). Additionally, several media were tested and MSCs osteogenic commitment in a non-osteogenic medium in the presence of ECs was carefully analyzed. The present results demonstrated that, independently of the relative cell ratio used, the coculture system promotes a significant increase in MSCs proliferation and differentiation. Furthermore, these results indicate that, for tissue engineering applications requiring a large number of MSCs committed into the osteogenic lineage, a coculture system with endothelial cells could be advantageous.
Results
Discussion A large number of MSCs committed to the osteogenic lineage is necessary for creating a tissue-engineered implant able to fill bone defects. It is generally accepted that MSCs play a critical role in bone tissue regeneration due to their differentiation capacity into distinct end-stage cell types, immunosuppressive properties and ability to produce a broad spectrum of bioactive macromolecules that are capable of establishing a regenerative microenvironment (Turnovcova et al., 2009). In bone marrow, MSCs represent a very small fraction of all nucleated cells: less than 0.01% (Bartmann et al., 2007; Lee & Park, 2009). As previously pointed out, this study focused on the use of a MSCs-endothelial cells coculture system as a strategy to modulate MSCs expansion and osteogenic differentiation in vitro. Our team is specifically interested in addressing this subject in the context of a minimally invasive bone regeneration strategy using injectable 3D matrices (Evangelista et al., 2007; Grellier et al., 2009c; Bidarra et al., 2010; Fonseca et al., 2011; Munarin et al., 2011). As reviewed by Grellier and colleagues (2009) there are some studies on the reciprocal regulation and functional relationship between bone and endothelial cells which, in turn, may be greatly influenced by the culture conditions (Grellier et al., 2009b). In a preliminary phase of the present study, and due to its impact in future cell studies, the appropriate medium for the coculture system was established. It is worth mentioning that, due to the dramatic influence that the batch of serum may have on MSCs behavior at different levels (proliferation, differentiation and gene expression) (Shahdadfar et al., 2005), a previous serum batch-selection was made and it was used to supplement all the tested media. Media selection was based on cell metabolic activity, total protein content and optical microscopy analyzes (Fig. 1). The tested media included those routinely used to culture the two cell types in monoculture (DMEM and M199 for MSCs and HUVECs, respectively) and also IMDM, a medium reported in several works for expansion and further coculture of both cell types (Guillotin et al., 2004; Villars et al., 2002; Grellier et al., 2009a; Guillotin et al., 2008; Villars et al., 2000; Meury et al., 2006). In several other studies, the medium selected for the cocultures was the same one used for ECs monocultures (Finkenzeller et al., 2006; Stahl et al., 2004; Wenger et al., 2004; Fuchs et al., 2009a; Fuchs et al., 2007; Fuchs et al., 2009b; Santos et al., 2009; Stahl et al., 2005; Unger et al., 2007; McEwen et al., 2003). Here, the medium selected was a DMEM+M199 mixture (1:1). After one week of culture, the cell performance in the presence of this medium was closer to the one obtained with DMEM and M199 for MSCs and HUVECs, respectively. Moreover, by choosing this mixed medium, cells in coculture will continue to be in contact with their expansion medium.