Nanotopographical cues mechanically regulate the secretion and osteoinductive potential of MSC‐derived exosomes for bone regeneration

Abstract The clinical use of mesenchymal stem cells (MSCs) is limited by potential biosafety concerns such as unwanted immune responses, positioning exosomes as a promising cell‐free alternative. However, enhancing exosome yield and functional cargo remains a key objective. Although biochemical and extracellular matrix‐associated biophysical cues have been implicated in regulating exosome secretion, how nanoscale organization of adhesive ligands through nanotopographical cues governs MSC exosome production and cargo composition remains largely unexplored. Herein, we engineered substrates with nanostructures of various sizes (200 or 700 nm) and densities (low or high) to spatially organize adhesive ligand presentation and modulate MSC exosome secretion through mechanosensing. In our findings, only the substrate with 700 nm nanostructures at low density (700 LD) boosts exosome secretion with ∼1.8‐fold increase compared to those on a flat surface. Mechanistically, 700 LD enhances integrin β1‐mediated focal adhesion formation and cell spreading, followed by Akt‐dependent Rab27a activation and enhanced exosome release, as supported by Akt inhibition and Rab27a knockdown/rescue experiments. Importantly, the cargo of the mechanoprimed exosomes promotes osteogenic differentiation of the MSCs in vitro and their osteoinductive potential for bone regeneration in vivo, in part through enrichment of miR‐548d‐5p. This study provides a strategic framework for engineering both MSC‐derived exosome production and therapeutic potency using customized nanotopographical cues, setting the stage for cell‐free regenerative therapies.