# The Role of Extracellular Matrix (ECM) Adhesion Motifs in Functionalised Hydrogels
## 6 Hydrogels Functionalised with Multiple Adhesion Motifs  
A combination of short peptide motifs may be suitable for creating biomimetic scaffolds, where differing concentrations of bioactive motifs can be used for functionalisation. This may help to create a biomimetic environment which more accurately represents what cells would experience in the native ECM, because in vivo, cells are exposed simultaneously to an array of ligand types, at various concentration gradients [130].  
PEG hydrogels have been modified with RGDS,IKVAV and YIGSR to create an environment capable of promoting nerve regeneration [130]. When all three bioactive ligands were used in combination, an additive effect on neurite extension was seen compared to RGDS alone, whereas all other pairwise combinations demonstrated an inhibitory effect or no effect whatsoever [130]. In a 2020 study, biocompatible hydrogels with improved cytotoxicity were produced using a combination of IKVAV and YIGSR peptides [131]. [PERSON] et al. [95] have also used a combination of RGD and N-cadherin to enhance differentiation potential using PLG hydrogels. Glycosaminoglycan production was shown to be significantly greater in dual peptide modified PLG, compared to PLG and PLG/RGD hydrogels [95]. It is evident that to create a biomimetic material, there may be advantages to seeding cells onto materials which allow simultaneous cell interaction with multiple bioactive ligands [130], but the combinations and number of ligands can show varying outcomes.  
Alongside the use of adhesion motifs in combination, some groups have reported that varying the density/concentration of adhesion motifs can affect cell behaviour. [PERSON] and [PERSON] [132] reported that dual functionalities hydrogels with the ligands RGD and YIGSR had a positive effect on PC12 adhesion and proliferation [132]. Good cell adhesion was observed on hydrogels containing equal concentrations of RGD and YIGSR. However, PC12 proliferation significantly increased as the density of RGD increased but YIGSR density remained unchanged [132]. Additionally, increasing YIGSR density in dual peptide hydrogels also had a positive effect on the production of the neuronal maker growth-associated protein 43 (GAP43), which in suggested promotion of neuronal differentiation [132]. [PERSON] and [PERSON], [133] cultured periodontal ligament stem cells (PDLSCs) in dual functionalised hydrogels containing RGD and GFOGER peptides to mimic the periodontal ECM [133]. More than 90% viability was maintained within all hydrogels tested [133]. ALP production and mineralisation were both enhanced by the presence of RGD and GFOGER when combined at varying concentrations; when high levels of RGD were paired with low-moderate levels of GFOGER, ALP activity increased by over half. Similarly, mineralisation increased by around 5-fold using moderate levels of RGD and high levels of GFOGER [133]. [PERSON] et al. [134] developed and characterised a synthetic polymer hydrogel poly (N\({}^{5}\)-(2-hydroxyethyl)-L-glutamine), modified with the integrin-binding motif RGD (PHEG-Tyr-RGD). Following cell encapsulation in PHEG-Tyr-RGD (6 mM) for 14 days, the cells demonstrated a normal MSC phenotype with a'spread' morphology. As the concentration of RGD was increased, cell-matrix and cell-cell interactions increased. Overall, this polymer hydrogel was found to be biocompatible, facilitating the adhesion and proliferation of MSCs, with interactions between the cells and the matrix itself influenced by RGD concentration [134].