Prechondrogenic ATDC5 cell attachment and differentiation on graphene foam; modulation by surface functionalization with fibronectin

Stephanie M Frahs, Jonathon C Reeck, Katie M. Yocham, Anders Frederiksen, Kiyo Fujimoto, Crystal M Scott, Richard S Beard, Raquel Brown, Trevor J Lujan, Ilia A. Solov'yov, David Estrada, Julia Thom Oxford
ACS Applied Materials and Interfaces

Graphene foam holds promise for tissue engineering applications. In this study, graphene foam was used as a three-dimensionscaffold to evaluate cell attachment, cell morphology, and molecular markers of early differentiation. The aim of this study was to determine if cell attachment and elaboration of an extracellular matrix would be modulated by functionalization of graphene foam with fibronectin, an extracellular matrix protein that cells adhere well to, prior to establishment of three-dimensional cell culture. Molecular dynamic simulation demonstrated that the fibronectin-graphene interaction was stabilized predominantly through interaction between the graphene and arginine side chains of the protein. Quasi-static mechanical testing indicated that fibronectin functionalization of graphene altered the mechanical properties of graphene foam. The elastic strength of the scaffold increased due to fibronectin, but the viscoelastic mechanical environment remained unchanged. A synergistic effect was observed in the elastic, equilibrium, and dynamic moduli in cultures where the graphene foam was coated with fibronectin. Cytoskeletal organization assessed by fluorescence microscopy demonstrated a fibronectin-dependent reorganization of actin cytoskeleton and an increase in actin stress fibers. Gene expression assessed by quantitative real time polymerase chain reaction of 9 genes encoding cell attachment proteins (Cd44,Ctnna1, Ctnnb1, Itga3 Itga5, Itgav, Itgb1, Ncam1, Sgce), 16 genes encoding extracellular matrix proteins (Col1a1, Col2a1, Col3a1, Col5a1, Col6a1, Ecm1, Emilin1 Fn1, Hapln1, Lamb3, Postn, Sparc, Spp1, Thbs1, Thbs2, Tnc), and 9 genes encoding modulators of remodeling (Adamts1, Adamts2, Ctgf, Mmp14 Mmp2, Tgfbi, Timp1, Timp2, Timp3) indicated that graphene foam provided a microenvironment conducive to expression of genes that are important in early chondrogenesis. Functionalization of graphene foam with fibronectin modified the cellular response to graphene foam, demonstrated by decreases in relative gene expression levels. These findings illustrate the combinatorial factors of microscale materials properties and nanoscale molecular features to consider in the design of three-dimensional graphene scaffolds for tissue engineering applications.