(83b) Bioactive Hydrogels Based On Collagen-Mimetic Proteins

Recent advances in adult mesenchymal stem cell (MSC) research have shown these cells to be a promising patient-derived cell source that are not only capable of extensive proliferation but also of differentiation into a range of cell types. Although significant advances in the use of MSCs for functional regeneration have been achieved, a number of obstacles remain before MSC-based engineered tissues can be considered viable clinical alternatives. In particular, rational design of scaffolds to elicit desired MSC lineage progression is problematic due to our incomplete understanding of MSC responses to extracellular matrix (ECM)-mediated stimuli, including matrix stiffness and biochemical cue identity. In the present work, we begin to address this challenge by probing MSC responses to collagen-based biochemical motifs using novel hybrid scaffolds. These hydrogels were generated by covalently crosslinking diacrylate-derivatized poly(ethylene glycol) (PEGDA) and a novel collagen-mimetic protein, termed Scl2-1. Scl2-1 is unique in that it contains the GXY repeats and stable triple helix of native collagen but lacks collagen's array of cell adhesion, cytokine binding, and enzymatic degradation sites. Thus, Scl2-1 provides a ?blank-slate? into which desired collagen adhesion sequences can be programmed by site-directed mutagenesis while maintaining the triple helical context natively associated with these motifs. The current work explores the impact of alpha1beta1 and alpha2beta1 integrin binding on focal adhesion kinase (FAK) and mitogen activated protein kinase (MAPK) signaling and associated MSC differentiation using modified Scl2-1. MSCs were encapsulated in PEGDA-Scl2 hydrogels containing various combinations of alpha1beta1 and alpha2beta1 integrin binding motifs (Scl2-2 - alpha1beta1 and alpha2beta1 site; Scl2-3 - alpha1beta1 site). In fabricating these gels, a weight ratio of PEGDA to Scl2 of 100:1 was used, implying that the elastic modulus, mesh size, and degradation rate of each hydrogel network would be dominated by PEGDA. Gels were cultured in media containing 10% FBS for 7 days. Over this time period, no alterations in gel volume or modulus were observed. Combined, these experimental conditions ensured that Scl2 identity was the primary design variable across gels. As expected, levels of active pFAK were significantly higher in the Scl2-2 and Scl2-3 gels than in the Scl2-1 negative control. In addition, the fraction of cells expressing myocardin (an early marker of smooth muscle cell differentiation) was substantially increased by the presence of Scl2-2 while expression of runx2 (an early osteoblast marker) was not (Fig. 1). Further examination of associated MAPK signaling suggested that pERK1/2 and p38 signaling played critical roles in the MSC fate decisions between smooth muscle and osteoblast lineages in these hydrogels. The present results demonstrate our ability to achieve a controlled 3D environment that can be used to probe MSC responses to highly defined collagen-based stimuli combinations while removing the influence of the array of additional signals provided by native collagen and while maintaining collagen's triple helical context. This controlled hydrogel platform enables more precise examination of the signaling underlying observed cell responses and should significantly enhance our understanding of the processes associated with MSC lineage progression.