br Material and methods br

Material and methods
Results
Discussion Differentiated chondrocytes are capable of proliferating and secreting numerous growth factors and cytokines to form the extracellular matrix in mature cartilage (Fischer et al., 2010; Keller et al., 2011). During chondrogenesis, these events are precisely regulated by different growth factors and soluble factors released from cartilage elements as well as from the perichondrium (Hwang et al., 2011). Previous studies have shown that chondrocyte-derived factors may influence the fate of mesenchymal NVP-TNKS656 via paracrine, juxtacrine or gap-junction signalling pathways (Cheng et al., 2009; Gelse et al., 2009), suggesting that these factors can stimulate the formation of extracellular matrix in precursor cells and could be essential for regenerative medicine applications (Hwang et al., 2008). These factors are, however, difficult to produce in sufficient quantity. The development of new molecules with increased activity in driving MSCs and ASCs towards chondrogenic differentiation is, therefore, a priority in regenerative medicine and pharmaceutics. On the other hand, there is accumulating evidence that co-culture multipotent stem cells, such as ASC and MSC, with patient derived chondrocytes can be used to induce chondrogenesis without the need for exogenous cytokines and growth factors (Wu et al., 2012a; Leijten et al., 2012). In addition, it has been shown that trophic factors secreted by the MSCs/ASCs induce cartilage formation by stimulating chondrocyte proliferation and matrix deposition by chondrocytes, rather than MSCs actively undergoing chondrogenic differentiation (Wu et al., 2011;, 2012b). Although these findings are interesting and could represent a suitable alternative for cartilage repair, we present here a different approach that is not based on the use of autologous chondrocytes. In this respect, we previously established a procedure that allowed us to generate a large quantity of BMP2/BMP6 heterodimer (BMP-2/6) by a chemical refolding method, and demonstrated that BMP-2/6 is a better inducer of differentiation of human embryonic stem cells (hESCs) than its homodimeric counterparts (Valera et al., 2010). In addition, using a segment-swapping RASCH strategy with BMP2 and Activin-A sequences, we have demonstrated that one such AB2 library chimera, AB208, exhibits the refolding characteristics of BMP2 while possessing Activin-like signaling attributes (Allendorph et al., 2011). Since BMP2 has been proven to enhance gene expression of cartilage-specific genes and proteoglycans in hMSCs (Sekiya et al., 2001, 2005), these chimeras can be good candidates for stem cell differentiation/guidance. In the present study we analyzed the chondrogenic potential of A/B2 chimera ligands on hASC. Firstly, we assayed their ability to up-regulate the expression of genes involved in chondrogenesis. AB2-ligands increased collagen II, aggrecan or Sox9 expression, which demonstrated a potential in inducing chondrogenic differentiation (Li et al., 2011; Yoo et al., 1998). The level of induction of chondrogenic genes was highly dependent on the chimeric ligands used, with AB235 showing the strongest chondrogenic potential both in monolayer and pellet culture systems. Time point evaluations confirmed that chondrogenic differentiation induced by AB235 exhibited a similar pattern to that of the cartilage maturation process with an initial increase of Col I and Sox9 and a decline at later times, a time-dependent increase in expression of Col II, and a marked decrease of Runx1 levels. Sox9 acts in early stages of chondrocyte differentiation, which directly induces type II collagen (Lefebvre et al., 1997) and is expressed in mesenchymal condensations (Lefebvre and de Crombrugghe, 1998). Decreased Col I gene expression suggested that AB2 ligands induced articular chondrocyte differentiation, as type-I collagen is either present in very small amounts or absent in hyaline cartilage, but abundant in fibrocartilage (Eyre and Muir, 1977; Roberts et al., 2009). Runx1 is a transcription factor highly expressed during early chondrogenesis and is downregulated in late stages of chondrocyte differentiation. Moreover, it has been demonstrated that Runx1 is capable of accelerating induction of MSC differentiation towards chondrogenesis by increasing the expression of early chondrocyte maturation markers (Wang et al., 2005). Thus, our results indicate that AB235 is superior in inducing chondrogenesis from early to late stages of maturation.