In our previous studies we successfully synthesized poly

In our previous studies, we successfully synthesized poly(hydroxyethyl methacrylate) (pHEMA) derived-polymers differing in the amount of 2-methacryloyloxyethyltrimethyl ammonium chloride (METAC) (Rosso et al., 2003; De Rosa et al., 2004). We demonstrated that positively charged moieties on the polymer surface influenced the substratum adhesion of primary human fibroblasts, as well as proliferation, triggering signals that regulated cell survival, purchase ryanodine progression, and expression of tissue-specific phenotypes. Although many technical applications and scientific developments have exploited Coulomb forces between charged polymers and oppositely charged biological macromolecules, fundamental aspects of these interactions are not well understood (Petrauskas et al., 2015). To date, there is very limited knowledge available on how charged polymers control cell-cell interactions and inter-cellular signal transduction. Cell-cell signaling pathways that lead to efficient differentiation of stem cells include the interaction of Ephrin ligands (ephrinB2) with Eph receptors (EphB4) (Nguyen et al., 2015; Gucciardo et al., 2014; Arthur et al., 2011). Receptor tyrosine kinases of the Eph family typically interact with the cell surface-associated ephrins at sites of cell–cell contact on neighboring cells (Pasquale, 2008). Eph–ephrin complexes lead to forward signal when Eph kinase activity propagate in the receptor-expressing cell, and reverse signal when tyrosine phosphorylation propagates in the ephrin-expressing cell. MSCs are self-renewing, multipotent progenitor cells, with multilineage potential to differentiate into adipocytes, osteocytes, and chondrocytes. In addition, MSCs can migrate to sites of inflammation, supporting hematopoiesis and homeostatic maintenance. Given their unique therapeutic properties, MSCs are a potential source of tissue repair (Squillaro et al., 2015).
Materials and methods
Results
Discussion This study demonstrates that a positive surface charge density affects cell–cell ephrinB2/EphB4 signaling modulating human MSC differentiation toward an osteoblast phenotype. The cell/material interaction is a complex, dynamic process in which the cell and the material synergistically influence the fate of the cell. Indeed, the specific physical properties of polymer surfaces (e.g., topography, charge, ζ-potential, and contact angle) can modulate the behavior of stem cells by inducing a cascade of events which start with cell adhesion and end with cell differentiation (Kaivosoja et al., 2012; Li et al., 2015). Several studies have recognized the surface cationic charge of polymers as an important co-factor in inducing stem cell differentiation toward osteoblasts and in stimulating bone formation, both in vitro and in vivo (Murphy et al., 2014; Tan et al., 2012). In particular, Iwai et al. synthetized materials with various charge ratios ranging from negative (−28mV) to positive (+21mV) able to induce drastic morphological changes in adipose-derived vascular progenitor cells (Iwai et al., 2013). Moreover, Zhang et al. designed positively-charged surface with tertiary amines with excellent cytocompatibility as well as remarkably upregulated osteogenesis-related gene/protein expressions and calcification of the bone marrow stem cells (Zhang et al., 2015). However, the role of the surface charge density on cell-cell communication and its effect on stem cell behavior are still unknown. Here, we synthetized two p(HEMA-co-METAC) polymers with high (pHM3) and low (pHM1) positive surface charge densities that differently modulate the MSCs osteogenic differentiation without affecting cell adhesion and proliferation. Our results demonstrated that the presence of high positive charge density perturbs ephrinB2/EphB4 interaction leading to an impairment in late-osteoblast differentiation. Receptor tyrosine kinases of the Eph family bind to cell surface-associated ephrin ligands on neighboring cells, generating bidirectional signals that affect both the receptor-expressing cells (forward signaling) and the ligand-expressing cells (reverse signaling) (Pasquale, 2008; Tonna et al., 2014). Several studies showed that ephrinB2/EphB4 signaling was implicated in the cell fate decisions of intestinal epithelial cells, hematopoietic lineages, and bone remodeling (Pasquale, 2010; Himanen et al., 2007; Pasquale, 2005; Arthur et al., 2009; Sancho et al., 2003). Zhao et al. demonstrated that the forward signaling through EphB4 enhances osteogenic differentiation in calvarial osteoblasts, suggesting that EphB4 is at the top of the regulatory cascade during osteoblast differentiation (Zhao et al., 2006). Although pHM1 and pHM3 non-significantly influenced ephrinB2 and EphB4 gene expression, phosphorylation of EphB4 was detected only in cells cultured on the low-charged material, confirming the activation of ephrinB2/EphB4 forward signaling. The role of the positive surface charge density in ephrinB2/EphB4 signaling, was confirmed using a blocking peptide, TNYL, which specifically binds to the ephrinB2 binding domain of the EphB4 receptor. This functional assay demonstrated that blocking the ephrinB2/EphB4 forward interaction impaired osteoblast differentiation also in MSCs cultured on pHM1. This finding suggests that positively charged polymers may influence stem cell behavior by perturbation of the Eph/ephrin interaction between neighboring cells, thus inhibiting both Eph/ephrin oligomerization in high-order clusters, and inter-cellular signal transduction (Himanen et al., 2010; Seiradake et al., 2010; Schaupp et al., 2014).