Osteogenic differentiation of mesenchymal stem cells in cell-laden culture of self-assembling peptide hydrogels

Mesenchymal stem cell (MSC) osteogenic differentiation requires scaffolds to support multiple stages of growth and differentiation signals. Fluorenyl-9-methoxycarbonyl diphenylalanine (Fmoc-FF) peptides self-assemble to create 3D nanofibers. Here, we cultured MSC in 2D and 3D Fmoc-FF layers to support their osteogenic differentiation. The stiffness of the hydrogels was tunable between 100 and 10,000 Pa which allows precise modulation of the cellular microenvironment. Scaffold stiffness impacted cell viability which softer scaffolds (100 Pa) favored higher viability. MSC formed spheroids in 3D hydrogel and showed spread morphology in 2D overlayers. Our results demonstrate that the Fmoc-FF 3D cultures significantly enhanced osteogenic differentiation, as evidenced by increased calcium deposition, elevated phosphatase activity, and the secretion of osteocalcin. We propose that the peptides provide integrin-binding sites that activate a cytoplasmic feedback loop essential for differentiation. These findings suggest that self-assembled Fmoc-FF peptide hydrogels, is a promising platform for bone tissue engineering applications.