# The Role of Extracellular Matrix (ECM) Adhesion Motifs in Functionalised Hydrogels
## 5 ECM-Derived Peptide Sequences and Their Use in Biomimetic Hydrogel Functionalisation
### Fibronectin: RGD Motif  
The tri-amino acid sequence RGD is the most extensively researched peptide adhesion motif. RGD serves as the main integrin-binding domain (residues 1615-1617 of fibronectin), and is found in many other ECM proteins such as vitronectin, collagen and bone sialoprotein [78]. In 1984, RGD motifs were first discovered as cell adhesion molecules, and the tripeptide sequence was identified as the minimal essential cell adhesion sequence of the ECM protein fibronectin [89]. The tendency for RGD to bind to an array of integrin receptors is thought to stem from the conformation of the loop containing RGD and the respective flanking amino acids [89]. In the literature, the relationship between RGD motifs within larger ECM proteins and cell attachment is evident. It has also been suggested that even small, synthetic peptides that contain the RGD sequence can promote cell attachment in a similar way to the much larger parent molecule fibronectin, highlighting the extent of its efficacy [90].  
Numerous groups have sought to imitate the roles of the ECM by functionalising hydrogels with the RGD motif. Human periosteum-derived cells (hPDCs) were encapsulated in PEG hydrogels functionalised with RGD, and results demonstrated the potential use of these hydrogels in articular cartilage regeneration to treat joint surface defects [91]. The hydrogels functionalised with RGD gave an average cell viability of approximately 85%, whereas cells cultured in hydrogels without RGD had viability of approximately 65%, decreasing to 40% after 4 weeks in culture [91]. A similar trend has also been identified elsewhere in the literature, where inclusion of the RGD peptide motif in various hydrogels can sustain a high cell survival rate [92; 93]. Corresponding to increased cell proliferation, DNA content in cells grown within hydrogels containing RGD was ten-fold higher than hydrogels without RGD (\(p<0.001\)) [91]. Other groups have similarly published cell viability, spreading and proliferation data which agree with the above results [94; 95; 96],providing evidence that RGD can be used as a biomimetic motif to replicate the adhesive characteristics of the ECM.  
The ability of commercial Vitrogel hydrogels with the RGD motif (VG-RGD), or without the RGD motif (VG-3D), to support the differentiation of human adipose stem cells (hASCs) has been investigated [97]. Following encapsulation, cells were evenly distributed throughout both hydrogels with high viability, indicating the promotion of a stable environment capable of cell adhesion and long-term cell support, much like the native ECM [97]. Moreover, hydrogels functionalised with RGD showed significantly enhanced differentiation of hASCs to a chondrogenic lineage [97], which has also been reported by other groups using different hydrogel types functionalised with RGD [98]. These results suggest that addition of RGD to hydrogel matrices can favour cell differentiation towards selected lineages. In relation to cartilage regeneration, the hydrogels were shown to have the functional capacity to replicate the complex characteristics of cartilaginous ECM, providing precise control of cellular functions to drive new tissue formation [97].