Nanostructured Hydrogel Surface Functionalization Controls C2C12 Myoblast Adhesion and Differentiation

Cell culture platforms benefit from chemical cues that recapitulate key aspects of the native biological environment. However, substantial challenges remain in designing scaffolds that also control the spatial organization of these cues. This is particularly difficult at gel interfaces, where functional groups must be presented within the outermost few nanometers to efficiently direct processes such as cell adhesion and spreading, but where nano- to microscale heterogeneities in synthetic hydrogel structure can obscure intended patterns. Here, we show that highly structured functional molecular layers, covalently transferred to polyacrylamide (PAAm), create hydrogel interfaces with defined surface chemistries, including amine, carboxylate, and zwitterionic groups, that regulate adhesion, proliferation, and differentiation of C2C12 murine myoblasts on hydrogels ranging from 10 to 90 kPa in stiffness. Functionalization with charged headgroup chemistries within this nanometer-thick interfacial layer increases cell density by 2-6-fold and average cell area by 50-100% relative to unfunctionalized PAAm, while increasing the abundance of extended triangular and rectangular cell morphologies by 5-30-fold. Upon differentiation, amine-functionalized 10 kPa surfaces exhibit geometric mean cell densities 24-fold higher than unfunctionalized surfaces, as well as ∼2-fold higher median numbers of nuclei per multinucleate structure.