Hybrid MPL Scaffolds with Nanoscale Mechanobiology for Bone-on-Chip

Engineering physiologically relevant 3D microenvironments is critical for studying cell behavior and advancing regenerative medicine. We present a new hybrid scaffold, fabricated via MultiPhoton Lithography (MPL), that integrates synthetic polymers (BisSR/CEA) with methacrylated collagen type I (Coll-MA) for single-cell enclosure and long-term culture. This is the first demonstration of a 3D MPL-printed biodegradable scaffold that mimics bone-like stiffness and allows spatially controlled, biodegradation-driven remodeling. The nanoscale feature size and mechanical properties are validated using Atomic Force Microscopy (AFM), while the nanoscale bioactivity of the scaffold is confirmed through Single-Molecule Localization Microscopy (SMLM). We track vinculin, a focal adhesion protein, with single-molecule resolution during mesenchymal stem cell (MSC) expansion and osteogenic differentiation. A new finding is time-dependent axial migration of vinculin clusters, independent of scaffold composition. Despite similar mechanosensing profiles, hybrid scaffolds significantly enhance osteogenic marker expression (collagen I, osteocalcin), revealing that scaffold bioactivity and geometry, not stiffness alone, direct stem cell fate. Cell expansion is highly dependent on scaffold composition, showing a biodegradation-driven remodeling over time. This platform offers a new tool to study cell-matrix interactions at the single-cell and single-molecule level and holds promise for Organ-on-Chip systems ( e . g . bone-cartilage interface models), and personalized regenerative therapies.