Hybrid Multiphoton Lithography Scaffolds for Nanoscale Mechanobiological Assessment in Microscale Bone Models

ABSTRACT We present a new hybrid 3D scaffold, fabricated via MultiPhoton Lithography (MPL), that integrates synthetic polymers (BisSR/CEA) with methacrylated collagen type I (Coll‐MA) and enables geometry‐dependent single‐cell confinement in most cages, while allowing cell–cell contact when the scaffold design permits multiple occupancy. We showcase that nanoscale structures with feature sizes down to hundreds of nanometers and locally tunable mechanical properties (kPa to MPa) can be achieved. Scaffold bioactivity is confirmed using 3D Single‐Molecule Localization Microscopy (SMLM). We quantified the dynamic 3D behavior of vinculin through its mechanoresponsive nanoscale localization. Notably, vinculin dynamics was independent of scaffold composition, including geometry, mechanical properties, and biodegradability. In contrast, collagen I and osteocalcin expression levels were strongly elevated in cells confined by hybrid scaffolds, indicating that scaffold bioactivity and geometry, rather than stiffness, govern stem cell fate. Our platform combines precisely tunable micro‐ and nanoscale environments mimicking extracellular matrix (ECM) features with super‐resolution imaging. This 3D tissue scaffold is component compatible with subsequent Organ‐on‐a‐Chip chamber integration and supports physiologically relevant bone tissue models.