Deciphering the Distinct Roles of Molecular and Supramolecular Chirality in Osteogenic Differentiation of Mesenchymal Stem Cells in 3D Hydrogels

Chirality is a pivotal determinant in stem cell differentiation, yet discerning the individual effects of chirality across different scales within native three-dimensional (3D) environments remains challenging. Here, a strategy is employed using nanostructures with controlled chirality to precisely assess the impact of molecular and supramolecular chirality on mesenchymal stem cell (MSC) osteogenic differentiation. We synthesized two pairs of enantiomers, l/d-phenylalanine (l/D-Phe) and l/d-1-naphthylalanine (L/D-1-Nap) derivatives, which could form four distinct chiral fibrous hydrogels with different molecular and supramolecular chiralities: L-^supP and D-^supP (^supP indicates supramolecular right-handed helix), and L-^supM and D-^supM (^supM denotes supramolecular left-handed helix). Both experimental and computational analyses reveal that the supramolecular ^supM/^supP helicity is governed by conformational changes in aromatic side chains, switching between outward and inward orientations. Intriguingly, MSCs encapsulated within these chiral fibers displayed osteogenic differentiation that was predominantly influenced by higher-order supramolecular chirality rather than molecular chirality. Specifically, ^supM-nanofibrils significantly promoted the MSC commitment to the osteoblast lineage, whereas ^supP-nanofibrils lacked this osteoinductive potential. Additionally, we observed subtle positive and negative modulations of MSC osteogenic differentiation by l- and d-enantiomeric molecular chiralities, respectively. Our study presents a strategy for chiral hydrogel design and delineates how supramolecular chirality surpasses molecular chirality in directing MSC osteogenesis within 3D hydrogels, highlighting the potential of chiral biomaterials in bone tissue engineering.