Effect of Graphene on Differentiation and Mineralization of Dental Pulp Stem Cells in Poly(4-vinylpyridine) Matrix in Vitro

We have investigated the influence of graphene nanoplatelet scaffolds for dental pulp cells (DPSCs) made from poly(4-vinylpyridine) (P4VP) either via spin-casting flat films or electrospinning nano- and microscale fibers. We found that graphene predominated over other factors in promoting differentiation of DPSCs. In the absence of graphene, real-time-polymerase chain reaction (RT-PCR) and energy dispersive X-ray (EDX) analyses indicated that the DPSCs differentiated along odontogenic lineages only on the nano- and microelectrospun scaffolds. Closer scanning electron microscopy (SEM) imaging revealed formation of banded collagen structures, which nucleated on the electrospun fibers in the absence of graphene. Biomineral deposition was templated on these fibers, with mineral to protein ratios similar to dentin. In the microfibers, the graphene was completely encapsulated and appeared to hinder biomineralization. Previously minimal biomineralization and banded collagen were observed on flat spun cast substrates. Addition of graphene appeared to induce nucleation of banded collagen fibers and template biomineral deposition. Addition of graphene did not affect the outcome of the DPSCs cultured on the nanofibers, which biomineralized regardless of graphene inclusion. Based on these results, we hypothesize that direct contact with graphene is the primary factor determining differentiation of the DPSCs. On the flat surface and nanoscale electrospun fibers, the graphene protrudes from the sample enabling direct contact with the extracellular matrix (ECM) and cells, while on the microfibers, the graphene is fully encapsulated within the matrix. TUNA imaging with scanning force microscopy showed enhanced conductivity on fibers with encapsulated graphene, which we hypothesize may change the conformation of adsorbed ECM proteins, affecting DPSCs differentiation.